Novel phosphoramidate compounds and methods of use

ABSTRACT

This invention provides compounds, compositions and methods for treating cancer, infectious disease, an autoimmune disorder or an inflammatory condition. Therapeutic compounds useful in the methods of this invention are 5′-phosphoramidatyl, 1,5-substituted pyrimidine compounds, derivatives, analogs and pharmaceutically acceptable salts thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 120 of U.S.Ser. No. 09/782,721, filed Feb. 12, 2001, which is a continuation ofU.S. Ser. No. 09/235,961, filed Jan. 22, 1999, now U.S. Pat. No.6,339,151B1, which in turn claims priority under 35 U.S.C. § 119(e) ofU.S. Provisional Application Nos. 60/072,264; 60/076,950; and60/108,634, filed Jan. 23, 1998, Mar. 5, 1998, and Nov. 16, 1998,respectively, now all abandoned. This application also claims priorityunder 35 U.S.C. § 120 of U.S. Ser. No. 09/856,127, filed Jul. 21, 1999,which in turn claims priority under 35 U.S.C. § 119(e) of U.S.Provisional Serial Nos. 60/145,356; 60/145,437; and 60/191,315, filedJul. 21, 1999, Jul. 22, 1999 and Mar. 21, 2000, respectively, now allabandoned. It also claims priority under 35 U.S.C. § 120 to U.S. Ser.No. 09/990,799, filed Nov. 16, 2001, which in turn claims priority under35 U.S.C. § 119(e) to U.S. Provisional Application No. 60/249,722, filedNov. 16, 2000, now abandoned. This application further claims priorityunder 35 U.S.C. § 120 to U.S. Ser. No. 10/051,320, filed Jan. 18, 2002,which in turn claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Serial No. 60/262,849, filed Jan. 19, 2001, now abandoned.The contents of all of the aforementioned applications are herebyincorporated by reference into the present disclosure.

TECHNICAL FIELD

[0002] The present invention is in the field of medicinal chemistry andrelates to other areas such as pharmacology, oncology and immunology. Inparticular, it provides compounds and methods to treathyperproliferative disorders.

BACKGROUND

[0003] Throughout and within this disclosure, various publications,patents, published patent applications and references are identified byfirst author and date, within parentheses, patent number, publicationnumber or by web address. If the complete bibliographic citation is notprovided after the publication or reference, it is at the end of thespecification, immediately preceding the claims. The disclosures of allpublications, references and information provided at the web addressesare hereby incorporated by reference into this disclosure to more fullydescribe the state of the art to which this invention pertains.

[0004] Hyperproliferative cells grow at a rate over that of normal orhealthy cells. The presence of these abnormal cells has been linked tomany pathologies, e.g., cancer, infectious disease, autoimmune disordersand inflammatory conditions or diseases. In many instances, they areuseful diagnostic indicators. In other instances, subcellular changeslinked to progression toward the hyperproliferative state are usefulprognostic indicators of disease progression or its curative treatment.

[0005] Cancer cells are hyperproliferative, i.e., characterized byuncontrolled growth, de-differentiation and genetic instability, thatexpress as aberrant chromosome number, chromosome deletions,rearrangements, loss or duplication beyond the normal diploid number.(Wilson, J. D. et al. (1991)). This genomic instability may be caused byseveral factors. One of the best characterized is the enhanced genomicplasticity which occurs upon loss of tumor suppressor gene function(e.g., Almasan, A. et al. (1995a) and Almasan, A. et al. (1995b)). Thegenomic plasticity lends itself to adaptability of tumor cells to theirchanging environment, and may allow for the more frequent mutation,amplification of genes, and the formation of extrachromosomal elements(Smith, K. A. et al. (1995) and Wilson, J. D. et al. (1991)). Thesecharacteristics provide for mechanisms resulting in more aggressivemalignancy because they allow tumors to rapidly develop resistance tonatural host defense mechanisms, biologic therapies (See Wilson, J. D.et al. (1991) and Shepard, H. M. et al. (1988)), as well as tochemotherapeutics (See Almasan, A. et al. (1995a); and Almasan, A. etal. (1995b)).

[0006] The heterogeneity of malignant tumors with respect to theirgenetics, biology and biochemistry as well as primary ortreatment-induced resistance to therapy mitigate against curativetreatment. Moreover, many anticancer drugs display only a low degree ofselectivity, causing often severe or even life threatening toxic sideeffects, thus preventing the application of doses high enough to killall cancer cells. Searching for anti-neoplastic agents with improvedselectivity to treatment-resistant pathological, malignant cellsremains, therefore, a central task for drug development.

[0007] The function of tumor suppressor genes is a major focus of recentattempts to develop innovative therapeutics for the treatment cancer.The products of tumor suppressor gene expression are generallycharacterized as negative regulators of cell proliferation (Knudson, A.G. (1993) and Weinberg, R. A. (1995)). Thus, therapeutic approaches todate include gene therapies to restore inactive or missing tumorsuppressor function in cancer cells to re-establish normal cellularfunction or induce apoptosis (Clayman, G. L. (2000) and Knudson, A. G.(1993)).

[0008] Loss of RB/p16 function can result in similar proinflammatory,proliferative and dedifferentiating effects on cells (Carson, R. A. andHaneji, N. (1999); Shim, J. et al. (2000); Wolff, B. and Naumann, M.(1999); DiCiommo et al. (2000)), and alteration in cell-cellinteractions (Plath et al. (2000)). Inactivation of tumor suppressorfunction by somatic mutation or via interaction with virally-encodedproteins is proposed to contribute to the proliferative/inflammatoryaspect of athersclerosis, restenosis or other hyperproliferativediseases (Tanaka, K. et al. (1999); Aoki, M. et al. (1999); Guevara, N.V. et al. (1999); and Iglesias, M. et al. (1998)). Finally, theexpression of the proinflammatory cytokine, macrophage inhibitory factor(MIF), may be capable of inactivating p53 function in some cell types(Hudson, J. D. et al. (1999); Cordon-Cardo, C. and Prives, C. (1999);and Portwine, C. (2000)).

[0009] Functional loss of tumor suppressor genes also has been linked toinflammatory or autoimmune diseases that have cellularhyperproliferation as one of their characteristics (Cordan-Cardo, C. andPrives, C. (1999)) and/or defective apoptosis (programmed cell death)(Mountz, J. D. et al. (1994)). These include: rheumatoid arthritis,systemic lupus erythmatosus, psoriatic arthritis, reactive arthritis,Crohn's disease, ulcerative colitis and scleroderma. Table 1 listsliterature examples which suggest that such a link may exist. TABLE 1Literature Examples Suggesting that Biological Expression of p53 TumorSuppressor Mutation/Inactivation Relates to Noncancer HyperproliferativeDisease, Autoimmune Disease and Inflammation. Impact Disease EffectReference Increased IL6 Proliferation Han, et al. (1999) InflammationRheumatoid Arthritis Increased metalloproteinases Tissue DegradationSun, Y. et al. (2000) Increased proliferation of Rheumatoid arthritisAupperle, K. R. et al. synovial cells (1998) Genetic instability Chronicinflammation Tak, P. P. et al. (2000) and disease progression Ulcerativecolitis Lang, S. M. et al. (1999) Increased expression of ProliferationBanerjee, D. et al. E2F regulated genes Drug resistance (1998) (TS,DHFR) Multiple autoimmune and inflammatory diseases Viral proteinsexpression Athersclerosis Tanaka, K. et al. leading to p53 inactivation(1999) Increased angiogensis Supports hyper-proliferative Zhang, L. etal. States, ex. enabling (2000) atheromaorpannus formation.

[0010] The hyperproliferative phenotype has also been linked toresistance to chemotherapy in cancer, infectious disease, autoimmunedisease and inflammatory conditions. Some hyperproliferative cellsoverexpress an intracellular enzyme that is related to any of a loss oftumor suppressor gene product function, drug resistance or geneticinstability. A number of cellular mechanisms are involved in drugresistance, e.g., altered metabolism of the drug, impermeability of thecell with regard to the active compound or accelerated drug eliminationfrom the cell, altered specificity of an inhibited enzyme, increasedproduction of a target molecule, increased repair of cytotoxic lesions,or the bypassing of an inhibited reaction by alternative biochemicalpathways. Enzymes activated or overexpressed and related to drugresistance include, but are not limited to thymidylate synthase (TS)(Lönn, U. et al. (1996); Kobayashi, H. et al. (1995); Jackman, A. L. etal. (1995)), dihydrofolate reductase (Banerjee, D. et al. (1995) andBertino, J. R. et al. (1996)), tyrosine kinases (TNF-α, Hudziak, R. M.et al. (1988)) and multidrug resistance (Stühlinger, M. et al. (1994));Akdas, A. et al. (1996); and (Tannock, I. F. (1996)); and ATP-dependentmultidrug resistance associated proteins (Simon, S. M. and Schnindler,M. (1994)). Alternatively, resistance to one drug may confer resistanceto other, biochemically distinct drugs. Amplication of certain genes isinvolved in resistance to chemotherapy. Amplification of dihydrofolatereductase (DHFR) is related to resistance to methotrexate whileamplification of the gene encoding thymidylate synthase is related toresistance to tumor treatment with 5-fluoropyrimidine.

[0011] Overexpression of enzymes encoded by human and animal pathogens,and in which the inhibitors have failed due to development ofresistance, also has been linked to disease. Indeed, resistance toantibiotics is a major health care problem. In infectious disease, mostdrug resistance is enzyme mediated. Typically, an enzyme expressed bythe infectious agent rapidly modifies the chemotherapeutic orantibiotic, thereby abolishing its therapeutic activitiy. Amplifiedexpression of beta-lactamases accounts for more than one-third of allbeta-lactam antibiotic resistant isolates (Felmingham and Washington(1999)), including the majority of resistant Haemophilis influenza(upper respiratory infections) and Moraxella catarrhalis (otitis media).In addition, genes conferring resistance to various alternative types ofantibiotics occur in nature and have become increasing common inpopulations of infectious organisms. Recently, infectious agentscarrying sets of genes simultaneously conferring resistance to multipleantibiotic agents have arisen making treatment by traditional antibiotictherapy difficult.

[0012] Thus, novel compounds and therapies are necessary overcome thelimitations of current therapies. This invention satisfies this need andprovides related advantages as well.

DISCLOSURE OF THE INVENTION

[0013] Novel phosphoramidatyl, 1,5-substituted pyrimidine compounds,derivatives, analogs, and pharmaceutically acceptable salts thereof andcompositions containing the compounds are provided by this invention.The compounds can be combined with an additional therapeutic drug ortherapy. The compounds and compositions are useful diagnostically andtherapeutically.

[0014] This invention provides methods for treating cells or tissueinvolved in a pathology characterized by hyperproliferative cells.Examples of pathologies include, but are not limited to cancer,infectious disease, autoimmune disease and an inflammatory condition.The cells and/or tissue are contacted with an effective amount of one ormore of a compound of this invention. Applicants have previously notedthat some compounds of this class are effective in treatinghyperproliferative disorders. See U.S. Pat. No. 6,339,151B1 and6,245,750, issued Jan. 15, 2002 and Jun. 12, 2001, respectively andpublished international patent applications PCT/US00/19844;PCT/US00/20007; and PCT/US00/20008.

[0015] The methods can be practiced in vitro, ex vivo and in vivo. Inone aspect, the cells or tissue are characterized by loss of tumorsuppressor function. In another aspect, the pathological mcellsoverexpress an endogenous intracellular enzyme such as thymidylatesynthase or a target enzyme. In yet a further aspect, the cells havebecome resistant to a chemotherapeutic drug, e.g., 5-fluorouracil (5FU).In another aspect, an infectious agent overexpresses a target enzymewhich in turn confers resistance.

[0016] When practiced in vivo, the invention provides a method fortreating a subject having a pathology characterized byhyperproliferative cells, e.g., cancer, an infectious disease,autoimmune disorder or an inflammatory condition, by delivering to thesubject an effective amount of at least one or more of the5′-phosphoramidatyl, 1,5-substituted pyrimidine, derivative, analog orpharmaceutically acceptable salt thereof. Methods for synthesizing thecompounds are described herein and in Applicants' prior patentliterature, e.g., PCT/US98/16607 and PCT/US99/01332, which describe thecompounds as “ECTA” compounds or prodrugs.

[0017] The methods are further useful to treat or ameliorate thesymptoms of a hyperproliferative disorder, e.g., cancer, infectiousdisease, autoimmune disease or an inflammatory condition, in a subjectby administering to the subject an effective amount of a compound orcomposition of this invention.

[0018] Further provided are compositions and methods for reversingreistance to a chemotherapeutic by contacting the resistant cells ortissue with a compound of this invention. Yet further provided aremethods and compositions for enhancing the efficacy of drugs that treator ameliorate symptons associated with cancer, infectious disease,autoimmune disease or an inflammatory condition.

[0019] Assays for identifying agents, therapies and combinations thereofthat inhibit the growth of pathological cells or tissue are alsoprovided herein.

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1 is a graph showing fluorescent products from incubation ofBromovinyl, 2′-Deoxyuridine Monophosphate (“BVdUMP”) with RecombinantHuman Thymidylate Synthase (“rHuTS”). Incubation of BVdUMP withthymidylate synthase (“TS”) results in a time and enzyme dependentgeneration of fluorescent product(s). BVdUMP was incubated with theindicated amounts of rHuTS in the standard reaction mixture at 30° C.(Materials and Methods), except that N5, N10-methylenetetrahydrofolatewas omitted from the reaction. The numbers adjacent to each data curverefer to TS enzyme units.

[0021]FIG. 2 shows the results of an experiment that demonstrates thatpreincubation with BVdUMP does not inactivate rHuTS. Human thymidylatesynthase was pre-incubated in reaction mixtures with and without 125 μMBVdUMP. After 20 hours, BVdUMP was added to a concentration of 125 μM,dUMP to a final concentration of 125 μM, and N5, N10-methylenetetrahydrofolate was added to 70 μM. Thymidylate synthase activity wasdetermined by measuring the increase in A₃₄₀. Solid circles(preincubated reaction), Open circles (no preincubation).

[0022]FIGS. 3A and 3B show detection of BVdUMP in H630R10 cells treatedwith NB1011. H630 R10 cells were treated with 100 μM NB1011 for 5 days,then analyzed by LC/MS as described in Materials and Methods.

[0023]FIG. 4 demonstrates that NB1011 does not irreversibly inactivateTS in vivo. The effect of NB1011 on TS activity in intact cells iscompletely reversible. TS activity was measured in intact RKO cells byrelease of [³H]₂O from 5-[³H]deoxyuridine as described in Materials andMethods. NB1011 was washed out of cells by replacing with fresh media,incubating for 60 minutes at 37 ° C., then repeating this procedure.Control and untreated cells were subjected to the same washingprocedure.

[0024]FIGS. 5A and 5B show that there are marked similarities between invitro efficacy requirements for NB1011 and anti-HER2. A), Data are takenfrom Tables 4, 5, and 8. B). Data from Shepard, et al. (1991). Verticalbars show standard error of means calculated using the Mann-Whitney Utest.

[0025]FIG. 6 shows that NB1011 is highly active against Tomudexresistant cancers. Cytotoxicity vs. TDX^(R) cell lines was measured inthe alamarBlue assay, as described in Materials and Methods, below.

[0026]FIG. 7 shows transcript levels of thymidylate synthase in humannormal and tumor colon tissues. RT-PCR analysis was performed asdescribed in Materials and Methods, below. The ratio of TS mRNA in tumorvs. normal tissue samples, each normalized to β-actin was (left toright) 14.35, 7.31, 0.75, 59.5, 2.53, 24.1, and 4.0.

[0027]FIG. 8A shows that NB1011 inhibits growth of 5-FU resistant coloncancer. Treatment of nude mice bearing H630R10 (5FU Resistant) humancolon carcinoma. Tumor measurements began on the first day of treatment(Day 1).

[0028]FIG. 8B shows long term response to NB1011. Analysis of pooleddata at Day 25. Statistical analysis is described in the Materials andMethods section below.

[0029]FIG. 9 is a graph showing mRNA levels of TS in multiple humantissues. TS mRNA levels were determined by using RT-PCR. The DNA bandscorresponding to thymidylate synthase were quantified and normalized tothat of β-actin by Molecular Dynamics Storm. Column 1 to 20 indicate theTS mRNA level in human normal tissues. The expression levels areexpressed as values relative to that of colon (column 16). Columns 21and 22 show the average TS transcript levels in 7 matched colon normaland cancer tissues. The expression values were relative to that ofnormal colon tissues (column 21).

MODES FOR CARRYING OUT THE INVENTION

[0030] General Techniques

[0031] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of organic chemistry,pharmacology, molecular biology (including recombinant techniques), cellbiology, biochemistry, and immunology, which are within the skill of theart. Such techniques are explained fully in the literature, such as,“MOLECULAR CLONING: A LABORATORY MANUAL” Second Edition (Sambrook etal., 1989); “OLIGONUCLEOTIDE SYNTHESIS” (M. J. Gait, ed., 1984); “ANIMALCELL CULTURE” (R. I. Freshney, ed., 1987); the series “METHODS INENZYMOLOGY” (Academic Press, Inc.); “HANDBOOK OF EXPERIMENTALIMMUNOLOGY” (D.M. Weir & C. C. Blackwell, eds.); “GENE TRANSFER VECTORSFOR MAMMALIAN CELLS” (J. M. Miller & M. P. Calos, eds., 1987); “CURRENTPROTOCOLS IN MOLECULAR BIOLOGY” (F. M. Ausubel et al., eds., 1987, andperiodic updates); “PCR: THE POLYMERASE CHAIN REACTION” (Mullis et al.,eds., 1994); “CURRENT PROTOCOLS IN IMMUNOLOGY” (J. E. Coligan et al.,eds., 1991); and J. March, ADVANCED ORGANIC CHEMISTRY: REACTIONS,MECHANISMS AND STRUCTURE, 4^(th) edition (John Wiley & Sons, NY (1992)).

[0032] Definitions

[0033] As used herein, certain terms may have the following definedmeanings.

[0034] As used in the specification and claims, the singular form “a,”“an” and “the” include plural references unless the context clearlydictates otherwise. For example, the term “a cell” includes a pluralityof cells, including mixtures thereof. Similarly, use of “a compound” fortreatment or preparation of medicaments as described herein contemplatesusing one or more compounds of this invention for such treatment orpreparation unless the context clearly dictates otherwise.

[0035] As used herein, the term “comprising” is intended to mean thatthe compositions and methods include the recited elements, but notexcluding others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination. Thus, a compositionconsisting essentially of the elements as defined herein would notexclude trace contaminants from the isolation and purification methodand pharmaceutically acceptable carriers, such as phosphate bufferedsaline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions of this invention.Embodiments defined by each of these transition terms are within thescope of this invention.

[0036] As used herein, the term “analog” is intended to mean astructural derivative of a compound that differs from it by at least oneelement. The term “derivative” is intended to mean a compound derived orobtained by another and containing the essential elements of the parentsubstance.

[0037] The term “alkyl” refers to and covers any and all groups whichare known as normal alkyl, branched-chain alkyl and cycloalkyl. As usedherein, “alkyl” is intended to include both branched, straight-chain,substituted or unsubstituted saturated aliphatic hydrocarbon groupshaving the specified number of carbon atoms. Examples of alkyl include,but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl, t-butyl, n-pentyl, and s-pentyl.

[0038] Lower alkyl means the above-defined broad definition of alkylgroups having 1 to 6 carbons in case of normal lower alkyl, and asapplicable 3 to 6 carbons for lower branch chained and cycloalkylgroups. Lower alkenyl is defined similarly having 2 to 6 carbons fornormal lower alkenyl groups, and 3 to 6 carbons for branch chained andcyclo- lower alkenyl groups. Lower alkynyl is also defined similarly,having 2 to 6 carbons for normal lower allynyl groups, and 4 to 6carbons for branch chained lower alkynyl groups.

[0039] “Haloalkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms, substituted with 1 or more halogen(for example —C_(V)F_(W) where v=1 to 3 and w=1 to (2v+1)). Examples ofhaloalkyl include, but are not limited to, trifluoromethyl,trichloromethyl, pentafluoroethyl, and pentachloroethyl.

[0040] “Carbocyclic” is intended to include saturated or unsaturatedring groups, such as cyclopropyl, cyclobutyl, or cyclopentyl. They maybe substituted or unsubstituted.

[0041] The term “alkenyl” refers to and covers normal alkenyl, branchchain alkenyl and cycloalkenyl groups having one or more sites ofunsaturation. Similarly, the term alkynyl refers to and coverssubstituted or unsubstituted normal alkynyl, and branch chain alkynylgroups having one or more triple bonds. “Alkynyl” is intended to includehydrocarbon chains of either a substituted or unsubstituted straight orbranched configuration and one or more triple carbon-carbon bonds whichmay occur in any stable point along the chain, such as ethynyl andpropynyl.

[0042] Some of the compounds of the present invention may have trans andcis (E and Z isomers. In addition, the compounds of the presentinvention may contain one or more chiral centers and therefore may existin enantiomeric and diasteromeric forms. Still further oxi and relatedcompounds of the present invention may exist in syn and anti isomericforms. The scope of the present invention is intended to cover all suchisomers per se, as well as mixtures of cis and trans isomers, mixturesof syn and anti isomers, mixtures of diastereomers and racemic mixturesof enantiomers (optical isomers) as well. In the present application,when no specific mention is made of the configuration (cis, trans, syn,anti, R or S) of a compound (or of an asymmetric carbon) then a mixtureof such isomers, or either one of the isomers is intended. In a similarvein, when in the chemical structural formulas of this application astraight line representing a valence bond is drawn to an as etriccarbon, then isomers of both R and S configuration, as well as theirmixtures are intended. Defined stereochemistry about an asymmetriccarbon is indicated in the formulas (where applicable) by a solidtriangle showing beta configuration, or by a hashed line showing alphaconfiguration.

[0043] All numerical designations, e.g., pH, temperature, time,concentration, and molecular weight, including ranges, areapproximations which are varied (+) or (−) by increments of 0.1. It isto be understood, although not always explicitly stated that allnumerical designations are preceded by the term “about”. It also is tobe understood, although not always explicitly stated, that the reagentsdescribed herein are merely exemplary and that equivalents of such areknown in the art.

[0044] A “subject” is a vertebrate, preferably a mammal, more preferablya human. Mammals include, but are not limited to, murines, simians,humans, farm animals, sport animals, and pets.

[0045] An “effective amount” is an amount sufficient to effectbeneficial or desired results. For example, a therapeutic amount is onethat achieves the desired therapeutic effect. This amount may be thesame or different from a prophylatically effective amount, which is anamount necessary to prevent onset of disease or disease symptoms. Aneffective amount can be administered in one or more administrations,applications or dosages.

[0046] A “pathological cell” is one that is pertaining to or arisingfrom disease. In one aspect, a pathological cell is identified fromnormal or healthy cells by presence of an endogenous intracellularenzyme such as TS or an activating enzyme. In another aspect, the celloverexpresses the enzyme (such as TS). In yet another aspect, the cellhas become resistant to prior drugs or therapy. In a further aspect, thecell has defective tumor suppressor function. In yet a further aspect,the expression of the activating enzyme occurs as a consequence ofinfection by a pathogenic organism.

[0047] Pathological cells can be hyperproliferative. A“hyperproliferative cell” means cells or tissue are dividing and growingat a rate greater than that when the cell or tissue is in a normal orhealthy state. Examples of such include, but are not limited to cancercells, cells associated with autoimmune or inflammatory conditions andcells associated with infectious disease, e.g., bacteria, parasites,virus, yeast, fungi, or plant or animal cells infected with an agent.Examples of viruses include but are not limited to Herpes, Varicellazoster, Hepatitis C and Epstein Barr virus. Examples of parasitesinclude but are not limited to T. brucei, T. cruzi, and Plasmodiumfalciparum. Examples of bacteria include, but are not limited to, allgram positive and gram negative bacteria, especially, Staphylococcus,sp., Enterococcus sp., Myoplasma sp., E. coli sp., Psudomonas sp.,Nisseria sp. In one embodiment, the infectious agents have becomeresistant to common antibiotics (see review by Murray, B. E. (1997)). Inother embodiments, the infectious agent expresses a “target enzyme” notexpressed by the host cell.

[0048] Hyperproliferative cells also include de-differentiated,immortalized, neoplastic, malignant, metastatic, and cancer cells suchas sarcoma cells, leukemia cells, carcinoma cells, or adenocarcinomacells. Specified cancers include, but are not limited to breast cancercells, hepatoma cells, liver cancer cells, pancreatic carcinoma cells,esophageal carcinoma cells, bladder cancer cells, gastrointestinalcancer cells, ovarian cancer cells, skin cancer cells, prostate cancercells, and gastric cancer cells.

[0049] In one aspect, hyperproliferative cells overexpress anintracellular enzyme that is related to any of a loss of tumorsuppressor gene product function, e.g. loss or inactivation ofretinoblastoma (RB) or p53, known to enhance expression of TS (Li, W. etal. (1995) or DHFR (Bertino, et al. (1996) and Li, W. et al. (1995)),drug resistance (e.g., amplification of the gene encoding TS is relatedto resistance to tumor treatment with 5-fluoropyrimidines), or geneticinstability or associated with a pathological phenotype. Alternatively,resistance to one drug may confer resistance to other, biochemicallydistinct drugs.

[0050] The enzyme glutathione-S-transferase was shown to be occasionallyelevated in some human tumors (Morgan, A. S. et al. (1998)), butnevertheless is excluded from an enzyme that is overexpressed as usedherein because it is a member of a gene family encoding enzymes withoverlapping specificities.

[0051] A “pathology characterized by hyperproliferative cells” includesbut is not limited to cancer, infectious disease, neoplasia, autoimmunedisorders and inflammatory conditions.

[0052] “Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson-Crick base pairing, Hoogstein binding, or inany other sequence-specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming amulti-stranded complex, a single self-hybridizing strand, or anycombination of these. A hybridization reaction may constitute a step ina more extensive process, such as the initiation of a PCR reaction, orthe enzymatic cleavage of a polynucleotide by a ribozyme.

[0053] Examples of stringent hybridization conditions include:incubation temperatures of about 25° C. to about 37° C.; hybridizationbuffer concentrations of about 6×SSC to about 10×SSC; formamideconcentrations of about 0% to about 25%; and wash solutions of about6×SSC. Examples of moderate hybridization conditions include: incubationtemperatures of about 40° C. to about 50° C.; buffer concentrations ofabout 9×SSC to about 2×SSC; formamide concentrations of about 30% toabout 50%; and wash solutions of about 5×SSC to about 2×SSC. Examples ofhigh stringency conditions include: incubation temperatures of about 55°C. to about 68° C.; buffer concentrations of about 1×SSC to about0.1×SSC; formamide concentrations of about 55% to about 75%; and washsolutions of about 1×SSC, 0.1×SSC, or deionized water. In general,hybridization incubation times are from 5 minutes to 24 hours, with 1,2, or more washing steps, and wash incubation times are about 1, 2, or15 minutes. SSC is 0.15M NaCl and 15 mM citrate buffer. It is understoodthat equivalents of SSC using other buffer systems can be employed.

[0054] A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) has a certain percentage (for example, 80%, 85%,90%, or 95%) of “sequence identity” to another sequence means that, whenaligned, that percentage of bases (or amino acids) are the same incomparing the two sequences. This alignment and the percent homology orsequence identity can be determined using software programs known in theart, for example those described in CURRENT PROTOCOLS IN MOLECULARBIOLOGY (F. M. Ausubel et al., eds., 1987) Supplement 30, section7.7.18, Table 7.7.1. Preferably, default parameters are used foralignment. A preferred alignment program is BLAST, using defaultparameters. In particular, preferred programs are BLASTN and BLASTP,using the following default parameters: Genetic code=standard;filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following Internet address:http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST.

[0055] “In vivo” gene delivery, gene transfer, gene therapy and the likeas used herein, are terms referring to the introduction of a vectorcomprising an exogenous polynucleotide directly into the body of anorganism, such as a human or non-human mammal, whereby the exogenouspolynucleotide is introduced to a cell of such organism in vivo.

[0056] The term “isolated” means separated from constituents, cellularand otherwise, in which the polynucleotide, peptide, polypeptide,protein, antibody, or fragments thereof, are normally associated with innature. For example, with respect to a polynucleotide, an isolatedpolynucleotide is one that is separated from the 5′ and 3′ sequenceswith which it is normally associated in the chromosome. As is apparentto those of skill in the art, a non-naturally occurring polynucleotide,peptide, polypeptide, protein, antibody, or fragments thereof, does notrequire “isolation” to distinguish it from its naturally occurringcounterpart. In addition, a “concentrated”, “separated” or “diluted”polynucleotide, peptide, polypeptide, protein, antibody, or fragmentsthereof, is distinguishable from its naturally occurring counterpart inthat the concentration or number of molecules per volume is greater than“concentrated” or less than “separated” than that of its naturallyoccurring counterpart. A polynucleotide, peptide, polypeptide, protein,antibody, or fragments thereof, which differs from the naturallyoccurring counterpart in its primary sequence or for example, by itsglycosylation pattern, need not be present in its isolated form since itis distinguishable from its naturally occurring counterpart by itsprimary sequence, or alternatively, by another characteristic such asglycosylation pattern. Although not explicitly stated for each of theinventions disclosed herein, it is to be understood that all of theabove embodiments for each of the compositions disclosed below and underthe appropriate conditions, are provided by this invention. Thus, anon-naturally occurring polynucleotide is provided as a separateembodiment from the isolated naturally occurring polynucleotide. Aprotein produced in a bacterial cell is provided as a separateembodiment from the naturally occurring protein isolated from aeukaryotic cell in which it is produced in nature.

[0057] “Host cell,” “target cell” or “recipient cell” are intended toinclude any individual cell or cell culture which can be or have beenrecipients for compounds or compositions of this invention, test agents,vectors or the incorporation of exogenous nucleic acid molecules,polynucleotides and/or proteins. It also is intended to include progenyof a single cell, and the progeny may not necessarily be completelyidentical (in morphology or in genomic or total DNA complement) to theoriginal parent cell due to natural, accidental, or deliberate mutation.The cells may be prokaryotic or eukaryotic, and include but are notlimited to bacterial cells, yeast cells, animal cells, and mammaliancells, e.g., murine, rat, simian or human.

[0058] A “subject” is a vertebrate, preferably an animal or a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.

[0059] A “control” is an alternative subject or sample used in anexperiment for comparison purpose. A control can be “positive” or“negative”. For example, where the purpose of the experiment is todetermine a correlation of the efficacy of a novel compound for thetreatment for a particular type of cancer, it is generally preferable touse a positive control (a compound known to exhibit the desiredtherapeutic effect) and a negative control (a subject or a sample thatdoes not receive the therapy or receives a placebo).

[0060] The terms “cancer,” “neoplasm,” and “tumor,” used interchangeablyand in either the singular or plural form, refer to cells that haveundergone a malignant transformation that makes them pathological to thehost organism. Primary cancer cells (that is, cells obtained from nearthe site of malignant transformation) can be readily distinguished fromnon-cancerous cells by well-established techniques, particularlyhistological examination. The definition of a cancer cell, as usedherein, includes not only a primary cancer cell, but also any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.When referring to a type of cancer that normally manifests as a solidtumor, a “clinically detectable” tumor is one that is detectable on thebasis of tumor mass; e.g., by such procedures as CAT scan, magneticresonance imaging (MRI), X-ray, ultrasound or palpation. Biochemical orimmunologic findings alone may be insufficient to meet this definition.

[0061] A neoplasm is an abnormal mass or colony of cells produced by arelatively autonomous new growth of tissue. Most neoplasms arise fromthe clonal expansion of a single cell that has undergone neoplastictransformation. The transformation of a normal to a neoplastic cell canbe caused by a chemical, physical, or biological agent (or event) thatdirectly and irreversibly alters the cell genome. Neoplastic cells arecharacterized by the loss of some specialized functions and theacquisition of new biological properties, foremost, the property ofrelatively autonomous (uncontrolled) growth. Neoplastic cells pass ontheir heritable biological characteristics to progeny cells.

[0062] The past, present, and future predicted biological behavior, orclinical course, of a neoplasm is further classified as benign ormalignant, a distinction of great importance in diagnosis, treatment,and prognosis. A malignant neoplasm manifests a greater degree ofautonomy, is capable of invasion and metastatic spread, may be resistantto treatment, and may cause death. A benign neoplasm has a lesser degreeof autonomy, is usually not invasive, does not metastasize, andgenerally produces no great harm if treated adequately.

[0063] Cancer is a generic term for malignant neoplasms. Anaplasia is acharacteristic property of cancer cells and denotes a lack of normalstructural and functional characteristics (undifferentiation).

[0064] A tumor is literally a swelling of any type, such as aninflammatory or other swelling, but modem usage generally denotes aneoplasm. The suffix “-oma” means tumor and usually denotes a benignneoplasm, as in fibroma, lipoma, and so forth, but sometimes implies amalignant neoplasm, as with so-called melanoma, hepatoma, and seminoma,or even a non-neoplastic lesion, such as a hematoma, granuloma, orhamartoma. The suffix “-blastoma” denotes a neoplasm of embryonic cells,such as neuroblastoma of the adrenal or retinoblastoma of the eye.

[0065] Histogenesis is the origin of a tissue and is a method ofclassifying neoplasms on the basis of the tissue cell of origin.Adenomas are benign neoplasms of glandular epithelium. Carcinomas aremalignant tumors of epithelium. Sarcomas are malignant tumors ofmesenchymal tissues.

[0066] One system to classify neoplasia utilizes biological (clinical)behavior, whether benign or malignant, and the histogenesis, the tissueor cell of origin of the neoplasm as determined by histologic andcytologic examination. Neoplasms may originate in almost any tissuecontaining cells capable of mitotic division. The histogeneticclassification of neoplasms is based upon the tissue (or cell) of originas determined by histologic and cytologic examination.

[0067] “Suppressing” tumor growth indicates a growth state that iscurtailed compared to growth without any therapy. Tumor cell growth canbe assessed by any means known in the art, including, but not limitedto, measuring tumor size, determining whether tumor cells areproliferating using a ³H-thymidine incorporation assay, or countingtumor cells. “Suppressing” tumor cell growth means any or all of thefollowing states: slowing, delaying, and “suppressing” tumor growthindicates a growth state that is curtailed when stopping tumor growth,as well as tumor shrinkage.

[0068] The term “culturing” refers to the in vitro propagation of cellsor organisms on or in media of various kinds. It is understood that thedescendants of a cell grown in culture may not be completely identical(morphologically, genetically, or phenotypically) to the parent cell. By“expanded” is meant any proliferation or division of cells.

[0069] An “autoimmune disorder” is any condition in which an organismproduces antibodies or immune cells which recognize the organism's ownmolecules, cells or tissues. Non-limiting examples of autoimmunedisorders include rheumatoid arthritis, Sjogren's syndrome, graft versushost disease, myasthenia gravis, and systemic lupus erythematosus.

[0070] An “inflammatory condition” shall mean those conditions that arecharacterized by a persistent inflammatory response with pathologicsequelae. This state is characterized by infiltration of mononuclearcells, proliferation of fibroblasts and small blood vessels, increasedconnective tissue, and tissue destruction. Chronic inflammatory diseasesinclude Crohn's disease, psoriasis, and asthma, are also included withinthe term “inflammatory condition.” Autoimmune diseases such asrheumatoid arthritis and systemic lupus erythematosus can also result ina chronic inflammatory state.

[0071] For the purpose of illustration only, treatment can be noted byreduction in the expression of an endogenous intracellular enzyme, e.g.,TS, reduction in the numbers of infectious agents, the reduction ininflammation, reduction of self-recognizing immune factors. In a furtheraspect, treatment is noted by the amelioration or reduction of symptomsof the disease, e.g., reduction in pathological cell growth or turnover,cachexia, tumor burden or elevated levels of immunological factorsassociated with a pathological or unhealthy state.

[0072] Treatment of arthritic conditions can result in decreased bloodvessel formation in cartilage, specifically joints, resulting inincreased mobility and flexibility in these regions. Treatment ofpsoriasis, administrationwill reduce dermatological symptoms such asscabbing, flaking and visible blood vessels under the surface of theskin.

[0073] In vitro treatment includes induction of apoptosis, as well asclinical (histological) and sub-clinical (e.g., biochemical and geneticchanges associated with a reversal or dimunition of the pathologicalstate.) Clinical and sub-clinical evidence of “treatment” will vary withpathology, the individual or subject, the cell or tissue type and thetreatment.

[0074] “An endogenous intracellular enzyme” is one that is expressed bythe cell whose regulation or expression can vary. In one aspect, theenzyme selectively activates a compound of this invention to produceproducts that confers treatment. In one aspect, the enzyme isoverexpressed in a diseased cell as compared to a normal healthy cell.An example of such is thymidylate synthase (TS).

[0075] The term “activating enzyme” as used herein means an enzyme thatis expressed by a pathogen in its native or natural environment. It isintended to distinguish enzymes or other agents that are administered toactivate a prodrug.

[0076] A “composition” is intended to mean a combination of active agentand another compound or composition, inert (for example, a solidsupport, a detectable agent or label) or active, such as an adjuvant.

[0077] A “pharmaceutical composition” is intended to include thecombination of an active agent with a carrier, inert or active, makingthe composition suitable for diagnostic or therapeutic use in vitro, invivo or ex vivo.

[0078] As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin, REMINGTON'SPHARMACEUTICAL SCIENCES, 15th Ed., Mack Publ. Co., Easton, Pa. (1975).

[0079] The Compounds

[0080] Therapeutic compounds for use in the methods of this inventionare one or more 5′-phosphoramidatyl 1,5-substituted pyrimidines,derivatives, analogs or pharmaceutically acceptable salts thereof. Thecompounds of this invention are nucleoside analogs comprising asubstituted or unsubstituted uracil base covalently joined to a sugarmodified by at least the addition of a 5′-phosphoramidate containing anamino acid residue. In one aspect, one or more of the compounds aresubstituted at the 5-position with a group that is extractable frompyrimidine by an endogenous, intracellular enzyme. The substituent atthe 1-position of uridine is selected from the group consisting ofsubstituted sugar, substituted thio-sugar, substituted carbocyclic,substituted cycloalkyl, and substituted acyclic substituents. Examplesof sugar groups include, but are not limted to, monosaccharide cyclicsugar groups such as those derived from oxetanes (4-membered ringsugars), furanoses (5-membered ring sugars), and pyranoses (6-memberedring sugars). Examples of furanoses include threo-furanosyl (fromthreose, a four-carbon sugar); erythro-furanosyl (from erythrose, afour-carbon sugar); ribo-furanosyl (from ribose, a five-carbon sugar);ara-furanosyl (also often referred to as arabino-furanosyl; fromarabinose, a five-carbon sugar); xylo-furanosyl (from xylose, afive-carbon sugar); and lyxo-furanosyl (from lyxose, a five-carbonsugar), and nucleoside analogs thereof.

[0081] Examples of thio sugar groups include the sulfur analogs of theabove sugar groups, in which the ring oxygen has been replaced with asulfur atom. Examples of carbocyclic groups include C₄ carbocyclicgroups, C₅ carbocyclic groups, and C₆ carbocyclic groups which mayfurther have one or more substituents, such as —OH groups.

[0082] Derivatives of the compounds of this invention include, forexample, “deoxy”, “keto”, and “dehydro” derivatives as well assubstituted derivatives. Derivatives also include salts, esters, andethers of the above compounds. Salts of the compounds of the presentinvention may be derived from inorganic or organic acids and bases.Examples of acids include hydrochloric, hydrobromic, sulfuric, nitric,perchloric, fumaric, maleic, phosphoric, glycollic, lactic, salicyclic,succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic andbenzenesulfonic acids. Other acids, such as oxalic, while not inthemselves pharmaceutically acceptable, can be employed in thepreparation of salts useful as intermediates in obtaining the compoundsof the invention and their pharmaceutically acceptable acid additionsalts. Examples of bases include alkali metal (e.g., sodium) hydroxides,alkaline earth metal (e.g., magnesium) hydroxides, ammonia, andcompounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl.

[0083] Examples of salts include: acetate, adipate, alginate, aspartate,benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate,camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate,pectinate, persulfate, phenylproprionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate and undecanoate. Otherexamples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group).

[0084] For therapeutic use, salts of the compounds of the presentinvention will be pharmaceutically acceptable. However, salts of acidsand bases which are non-pharmaceutically acceptable may also find use,for example, in the preparation or purification of a pharmaceuticallyacceptable compound.

[0085] Esters of the compounds identified by the method of thisinvention include carboxylic acid esters (i.e., —O—C(═O)R) obtained byesterification of the 2′-, 3′- and/or 5′-hydroxy groups, in which R isselected from (1) straight or branched chain alkyl (for example,n-propyl, t-butyl, or n-butyl), alkoxyalkyl (for example,methoxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (forexample, phenoxymethyl), aryl (for example, phenyl optionallysubstituted by, for example, halogen, C₁₋₄alkyl, or C₁₋₄alkoxy oramino); (2) sulfonate esters, such as alkylsulfonyl (for example,methanesulfonyl) or aralkylsulfonyl; (3) amino acid esters (for example,L-valyl or L-isoleucyl); (4) phosphonate esters and (5) mono-, di- ortriphosphate esters. The phosphate esters may be further esterified by,for example, a C₁₋₂₀ alcohol or reactive derivative thereof, or by a2,3-di-(C₆₋₂₄)acyl glycerol. In such esters, unless otherwise specified,any alkyl moiety present advantageously contains from 1 to 18 carbonatoms, particularly from 1 to 6 carbon atoms, more particularly from 1to 4 carbon atoms. Any cycloalkyl moiety present in such estersadvantageously contains from 3 to 6 carbon atoms. Any aryl moietypresent in such esters advantageously comprises a phenyl group. Examplesof lyxo-furanosyl compound derivatives of the present invention include,for example, those with chemically protected hydroxyl groups (e.g., withO-acetyl groups), such as 2′-O-acetyl-lyxo-furanosyl;3′-O-acetyl-lyxo-furanosyl; 5′-O-acetyl-lyxo-furanosyl;2′,3′-di-O-acetyl-lyxo-furanosyl and2′,3′,5′-tri-O-acetyl-lyxo-furanosyl.

[0086] Ethers of the compounds of the present invention include methyl,ethyl, propyl, butyl, isobutyl, and sec-butyl ethers.

[0087] Compounds useful in the methods of this invention can bedescribed as the L and D isomers of compounds having one of thefollowing structures:

[0088] or tautomers thereof, wherein in Formula C, R¹² or R¹³ may be thesame or different and are selected from the group consisting of oxo, OHor NHNH₂, wherein a is 0 or 1, providing that if a is 0 and R¹³ is oxo,then a double bond exits between position 3 and 4 and R¹² is NHNH₂;further providing that if a is 0 and R¹² is oxo, then a double bondexists between position 2 and 3 and R¹³ is NHNH₂; further providing thatif a is 1, then R¹² and R¹³ are both oxo.

[0089] While not wishing to be bound by any theory, in one aspect of theabove formulae (A, B and C), R¹ (at the 5-position) is or contains aleaving group which is a chemical entity that has a molecular dimensionand electrophilicity compatible with extraction from the pyrimidine ringby an endogenous, intracellular enzyme (e.g., thymidylate synthase). Anembodiment for the substituent in the R¹ position is one that couldundergo an allylic interchange.

[0090] Another example is an alkenyl group of the formula, i.e.,(—CH═CH)_(n)—R⁴, wherein n is 0 or an integer from 1 to 10, and R⁴ is ahalogen such as I or Br, CN or mercury, or alternatively, R¹ is orcontains a group selected from hydrogen, alkyl, alkene, alkyne, hydroxy,—O-alkyl,—O-aryl, O-heteroaryl, —S-alkyl, —S-aryl, a cyanide, cyanate,thiocyanate halovinyl group, halomercuric group, —S-heteroaryl, —NH₂,—NH-alkyl, —N(alkyl)₂, —NHCHO, —NHOH, —NHO-alkyl, NH₂CONHO—, and NHNH₂.For example, when n is 0 or an integer from 1 to 10, R⁴ is CH₂-O—A,wherein A is a

[0091] phosphoramide derivative, or a compound of the formula:

[0092] Alternatively, in the above formulae (A, B or C), R¹ can be amoiety of the formula:

[0093] wherein, R⁴ is a toxophore.

[0094] In one aspect of Formula D, R² is or contains a divalent electronconduit moiety. In one embodiment, R² is or contains a mono- orpolyunsaturated electron conduit acting to conduct electrons away fromthe pyrimidine ring and toward R⁴. In another embodiment, R² is selectedfrom the group consisting of an unsaturated hydrocarbyl group, anaromatic hydrocarbyl group comprising one or more unsaturatedhydrocarbyl groups, and a heteroaromatic group comprising one or moreunsaturated hydrocarbyl groups.

[0095] In a yet further aspect, m is 0 and R² is selected from the groupconsisting of:

[0096] wherein R⁵ is independently the same or different and is selectedfrom the group consisting of a linear or branched alkyl group havingfrom 1 to 10 carbon atoms, a cycloalkyl group having from 3 to 10 carbonatoms, CN and a halogen.

[0097] In one embodiment of Formula D, R² is an unsaturated hydrocarbylgroup having a structure selected from the group consisting of:

[0098] In another embodiment of Formula D, R² is an aromatic hydrocarbylgroup having a structure selected from the group consisting of:

[0099] In yet another embodiment of Formula D, R² is a heteroaromaticgroup having a structure selected from the group consisting of:

[0100] wherein J is a heteroatom, such as —O—, —S—, or —Se—, or aheteroatom group, such as —NH— or —NR^(ALK)—, where R^(ALK) is a linearor branched alkyl having 1 to 10 carbon atoms or a cycloalkyl grouphaving 3 to 10 carbon atoms.

[0101] In an alternative embodiment of Formula D, R³ is a divalentspacer moiety, also referred to as a spacer unit. Divalent spacersinclude, but are not limited to, a moiety having a structure:

[0102] wherein R⁵ is the same or different and is independently a linearor branched alkyl group having from 1 to 10 carbon atoms, or acycloalkyl group having from 3 to 10 carbon atoms.

[0103] In an alternative aspect of Formula D, R³ is a divalent spacermoiety having a structure selected from the group consisting of:

[0104] In yet another aspect of Formula D, R² and R³, taken togetherform a structure selected from the group consisting of:

[0105] In one embodiment, R⁴ (R⁴ in Formula D or R¹ in Formulae A, B orC) is or contains a leaving group that is activated or released by anintracellular enzyme. In one embodiment, R⁴ is or contains a grouphaving a structure selected from the group consisting of F, Cl, Br, I,CN, SO₃H, CO₂H, CO₂CH₂CH₃, CO₂CH₃, SI(CH₃)₃, CHO, NO₂, CF₃, CCl₃,CH═C(R¹⁵)₂ and a derivative of cisplatin, such as:

[0106] or a substituent selected from the structures:

[0107] wherein X_(a) and X_(b) are independently the same or differentand are selected from the group consisting of Cl, Br, I, and a potentleaving group and wherein Y_(a), Y_(b) or Y_(c) are independently thesame or different and are hydrogen or F and wherein Z, Z_(a) and Z_(b)are independently the same or different and are selected from the groupconsisting of O and S; and with respect to Formula C, R¹⁴ is hydrogen orF, providing if R¹⁴ is F, then a is 1 and R¹² and R¹³ are both oxo.

[0108] Q is a 5′ phosphoramidate derivative, analog or pharmaceuticallyacceptable salt of a sugar as defined above, e.g., Q is selected fromthe group consisting of:

[0109] In the above Formula F, R₂ and R₃ are independently the same ordifferent and are selected from the group consisting of Br, Cl, F, I, H,OH, OC(═O)CH₃, —O-and —O—Rg, wherein Rg is a hydroxyl protecting groupother than acetyl. R₇ is attached to Q at the 5′ position of Q and is anamino acid containing phosphoramidate group. Any of the members ofFormulae F may be in any enantiomeric, diasteriomeric, or stereoisomericform, including D-form, L-form, α-anomeric form, and β-anomeric form.

[0110] In a specific embodiment, Q has the formula:

[0111] wherein R₂ and R₃ are independently the same or different and areindependently H, —OH, —OC(═O)CH₃, or —O-Rg, wherein Rg is a hydroxylprotecting group other than acetyl. R₇ is as defined above.

[0112] In a further specific embodiment, Q has the following structure:

[0113] In each of Formulae F, G, or H, R₇ is a phosphoramidate groupderived from an amino acid, including, for example, the twenty naturallyoccurring amino acids, e.g., alanine and tryptophan. In one embodiment,R₇ is a phosphoramidate group derived from tryptophan, for example agroup having the structure:

[0114] The above group, and methods for its preparation, are describedin Abraham et al., (1996).

[0115] In another embodiment, R₇ is or contains a group having thestructure:

[0116] wherein R₈ is a side chain of any amino acid, its derivative, itsanalogue or its isomer and wherein R₉ is selected from the groupconsisting of hydrogen, an unbranched or branched acyclic alkyl groupcontaining from 1 to 5 carbons, a cyclic saturated alkyl groupcontaining from 3 to 8 carbons, an aryl group and an adamantyl group. Inone aspect, R₈ is a side chain of any amino acid, its derivative, itsanalogue or its isomer with the proviso that when R₈ is alanine, R₉ isnot selected from the group consisting of methyl, ethyl,methyl-tert-butyl, iso-propyl, methyl-cyclopropyl, cyclohexyl andbenzyl. In a further aspect, R₈ is a side chain of any amino acid, itsderivative, its analogue or its isomer with the proviso that when R₉ ismethyl, R₈ is not selected from the group consisting of tryptophan,valine, glycine, leucine, phenylalanine and aspartic acid. In a furtheraspect, R₈ is a side chain of alanine and R₉ is selected from the groupconsisting of benzyl, methyl-cyclopropyl, cyclohexyl, iso-propyl,methyl-tert-butyl, cycloheptyl, cyclooctyl and methyl-adamantyl. In afurther aspect, R₈ is a side chain of alanine and R₉ is selected fromthe group consisting of cycloheptyl, cyclooctyl and methyl-adamantyl. Ina further aspect, R₈ is a side chain of tryptophan and R₉ is methyl.

[0117] In another aspect, R₈ and R₉ are defined as above, wherein thenucleoside or its analog thereof is 2′-deoxy. In one aspect, when R₈ isalanine and R₉ is methyl, then R₁ is not bromovinyl. In another aspect,when R₈ is alanine, R₉ is methyl and R₁ is not bromovinyl, then thenucleoside or analog is not 2′-deoxy.

[0118] In one embodiment, R₇ is a phosphoramidate group derived fromalanine, e.g., a group having the structure:

[0119] The above group, and methods for its preparation, are describedin McGuigan et al. (1993) and McGuigan et al. (1996).

[0120] In further embodiments, R₇ is or contains a substituent selectedfrom the group consisting of:

[0121] It is intended, although not always stated that the compounds ofthis invention may be in any enantiomeric, diasteriomeric, orstereoisomeric form, including, D-form, L-form, α-anomeric form, andβ-anomeric forms. The compounds may be in a salt form, or in a protectedor prodrug form, or a combination thereof, for example, as a salt, anether, or an ester.

[0122] In a further aspect, R⁷ is as defined above and R₁ is abromovinyl group or a group shown in Table 2, below.

[0123] Specific compounds having the L or D structures are shown inTable 2, below. Compounds are identified by structure and a numericaldesignation. TABLE 2

R₁

NB 1011

NB 1012

NB 1013 —CF₃ NB 1014

NB 1016

NB 1017 —≡—SiMe₃ NB 1018 —≡—H NB 1019 —≡—C₈H₁₇ — —C₈H₁₇ —

[0124] Further embodiments of compounds of this invention are providedbelow.

[0125] wherein X_(d) and X_(e) are independently the same or differentand are selected from the group consisting of Cl, Br, I, and CN. In amore preferred aspect, X_(d) is Cl or Br and X_(e) is hydrogen.

[0126] A compound having the structure:

[0127] wherein X_(f) and X_(g) are independently the same or differentand are selected from the group consisting of Cl, Br, I, and CN. In apreferred embodiment, X_(f) and X_(g) are the same and are each is Cl orBr.

[0128] A compound having the structure of the formula:

[0129] wherein X_(h) and X_(i) are independently the same or differentand are selected from the group consisting of Cl, Br, I, and CN. In apreferred embodiment, X_(h) and X_(l) are independently the same ordifferent and are Cl or Br and in a more preferred embodiment, X_(h) andX_(l) are both Br.

[0130] A compound having the structure:

[0131] wherein R⁸ is a lower straight or branched chain alkyl.

[0132] A compound having the structure:

[0133] wherein R⁸ and R⁹ are lower straight or branched chain alkyls andR¹⁰ is hydrogen or CH₃.

[0134] A compound having the structure:

[0135] wherein R¹⁰ is hydrogen or CH₃.

[0136] A compound having the structure:

[0137] wherein X is selected from the group consisting of CO₂Et, Cl, andBr.

[0138] A compound having the structure:

[0139] wherein R₁ is a side chain of any naturally occurring amino acid,its analogue or its isomer; and wherein R₂ is selected from the groupconsisting of hydrogen, an unbranched or branched acyclic alkyl groupcontaining from 1 to 5 carbons, a cyclic saturated alkyl groupcontaining from 3 to 8 carbons and a benzyl group and itspharmaceutically acceptable salts.

[0140] A compound having the structure:

[0141] The compounds can be combined with a carrier, such as apharmaceutically acceptable carrier, for use in vitro and in vivo.

[0142] Formulations for In Vivo Administration

[0143] While it is possible for the composition ingredient to beadministered alone, it is preferable to present it as a pharmaceuticalformulation comprising at least one active ingredient, as defined above,together with one or more pharmaceutically acceptable carriers thereforeand optionally other therapeutic agents. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient.

[0144] Formulations of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets, each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. The active ingredient may also bepresented a bolus, electuary or paste.

[0145] A tablet may be made by compression or molding, optionally withone or more accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g., povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g., sodiumstarch glycolate, cross-linked povidone, cross-linked sodiumcarboxymethyl cellulose) and/or surface-active or dispersing agent.Molded tablets may be made by molding in a suitable machine a mixture ofthe powdered compound moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and may be formulated so asto provide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile. Tablets may optionally beprovided with an enteric coating, to provide release in parts of the gutother than the stomach.

[0146] Formulations suitable for topical administration in the mouthinclude lozenges comprising the active ingredient in a flavored basis,usually sucrose and acacia or tragacanth; pastilles comprising theactive ingredient in an inert basis such as gelatin and glycerin orsucrose and acacia; and mouthwashes comprising the active ingredient ina suitable liquid carrier.

[0147] Pharmaceutical compositions for topical administration accordingto the present invention may be formulated as an ointment, cream,suspension, lotion, powder, solution, past, gel, spray, aerosol or oil.Alternatively, a formulation may comprise a patch or a dressing such asa bandage or adhesive plaster impregnated with active ingredients andoptionally one or more excipients or diluents.

[0148] For diseases of the eye or other external tissues, e.g., mouthand skin, the formulations are preferably applied as a topical ointmentor cream containing the active ingredient in an amount of, for example,about 0.075 to about 20% w/w, preferably about 0.2 to about 25% w/w andmost preferably about 0.5 to about 10% w/w. When formulated in anointment, the composition may be employed with either a paraffinic or awater-miscible ointment base. Alternatively, the ingredients may beformulated in a cream with an oil-in-water cream base.

[0149] If desired, the aqueous phase of the cream base may include, forexample, at least about 30% w/w of a polyhydric alcohol, i.e., analcohol having two or more hydroxyl groups such as propylene glycol,butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycoland mixtures thereof. The topical formulations may desirably include acompound that enhances absorption or penetration of the ingredientsthrough the skin or other affected areas. Examples of such dermalpenetration enhancers include dimethylsulfoxide and related analogs.

[0150] The oily phase of the emulsions of this invention may beconstituted from known ingredients in a known manner. While this phasemay comprise merely an emulsifier (otherwise known as an emulgent), itdesirably comprises a mixture of at least one emulsifier with a fat oran oil or with both a fat and an oil. Preferably, a hydrophilicemulsifier is included together with a lipophilic emulsifier that actsas a stabilizer. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabilizer(s) make up theso-called emulsifying wax, and the wax together with the oil and/or fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

[0151] Emulgents and emulsion stabilizers suitable for use in theformulation of the present invention include Tween 60, Span 80,cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodiumlauryl sulfate.

[0152] The choice of suitable oils or fats for the formulation is basedon achieving the desired cosmetic properties, since the solubility ofthe active compound in most oils likely to be used in pharmaceuticalemulsion formulations is very low. Thus, the cream should preferably bea non-greasy, non-staining and washable product with suitableconsistency to avoid leakage from tubes or other containers. Straight orbranched chain, mono- or dibasic alkyl esters such as di-isoadipate,isocetyl stearate, propylene glycol diester of coconut fatty acids,isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters known asCrodamol CAP may be used, the last three being preferred esters. Thesemay be used alone or in combination depending on the propertiesrequired. Alternatively, high melting point lipids such as white softparaffin and/or liquid paraffin or other mineral oils can be used.

[0153] Formulations suitable for topical administration to the eye alsoinclude eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent for theingredients. The ingredients are preferably present in such formulationin a concentration of about 0.5 to about 20%, advantageously about 0.5to about 10%, particularly about 1.5% w/w.

[0154] Formulations for rectal administration may be presented as asuppository with a suitable base comprising, for example, cocoa butteror a salicylate.

[0155] Formulations suitable for vaginal administration may be presentedas suppositories, tampons, creams, gels, pastes, foams or sprayformulations containing in addition to the ingredients, such carriers asare known in the art to be appropriate.

[0156] Formulations suitable for nasal administration, wherein thecarrier is a solid, include a coarse powder having a particle size, forexample, in the range of about 20 to about 500 microns which isadministered in the manner in which snuff is taken, i.e., by rapidinhalation through the nasal passage from a container of the powder heldclose up to the nose. Suitable formulations wherein the carrier is aliquid for administration as, for example, nasal spray, nasal drops, orby aerosol administration by nebulizer, include aqueous or oilysolutions of the ingredients.

[0157] Formulations suitable for parenteral administration includeaqueous and non-aqueous isotonic sterile injection solutions which maycontain antioxidants, buffers, bacteriostats and solutes which renderthe formulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. The formulations may be presented in unit-dose ormulti-dose sealed containers, for example, ampoules and vials, and maybe stored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules andtablets of the kind previously described.

[0158] It should be understood that in addition to the ingredientsparticularly mentioned above, the formulations of this invention mayinclude other agents conventional in the art having regard to the typeof formulation in question, for example, those suitable of oraladministration may include such further agents as sweeteners, thickenersand flavoring agents.

[0159] Compositions of the formula of the present invention may also bepresented for the use in the form of veterinary formulations, which maybe prepared by methods that are conventional in the art.

[0160] Methods of Treatment

[0161] Pathological cells, tissues and pathologies characterized byhyperproliferative cells are treated by contacting the cells or tissueassociated with these pathologies with an effective amount of a compoundof this invention. The contacting can be any one or more of in vitro, exvivo and in vivo.

[0162] When practiced in vivo in a subject other than a human patientsuch as a mouse, the method provides an animal model for use indiscovering alternative agents and therapies. In a human patient, themethod treats pathologies characterized by hyperproliferative cells,e.g., cancer, infectious disease, an autoimmune disorder or inflammatorycondition. Methods for detecting clinical and sub-clinical evidence ofeffective therapy are known in the art and described herein. In each ofthese methods, an effective amount of a compound of this invention isdelivered or administered to the subject, e.g., mouse or human patient.

[0163] In one aspect, this invention is directed to methods forinhibiting the proliferation or growth of an infectious agent or a cellinfected with the agent by contacting the agent or infected cell with acompound of this invention. The methods and compositions of thisinvention are useful to preferentially inhibit the growth orproliferation of cells that express or contain activating enzyme, forexample microbial cells, virally infected cells or cells infected withother pathogens. Overexpression of the enzyme is not required, asspecificity is related to the species-specificity of the compound to theactivating enzyme expressed by the pathogen. The activating enzyme mayor may not be expressed by the host cell. However, even if the cellexpresses its own version of the enzyme, the compound is selective onthe basis that it is preferentially activated by the version of theenzyme expressed by the infectious agent as compared to the version ofthe enzyme expressed by the host cell. The activating enzyme can be thewild-type or a mutated version which has developed resistance to priorart therapeutics (Hooker, et al. (1996)).

[0164] Examples of activating enzymes that are selective targets for thecompounds and methods of this invention include, but are not limited to,thymidylate synthase (TS), dihydrofolate reductase (DHFR) andβ-lactamase activating enzymes.

[0165] The concepts of this invention are illustrated using theactivating enzyme thymidylate synthase and its expression in human tumorcells. However, the use of TS is merely illustrative and the claims arenot to be construed as limited to systems which target TS. Thymidylatesynthase was used herein as the target,. activating enzyme because ofthe high degree of characterization of its structure and function(Carreras and Santi (1995)), the fact that it is encoded by a singlegene, not a gene family (compare for example the family of enzymes notedas glutathione-S-transferase (GST)). In addition, TS overexpression isthe result of acquired resistance to chemotherapeutics. Similarly, inone embodiment, the activating enzyme can be expressed as a result ofresistance to prior therapy.

[0166] Other target activating enzymes include, but are not limited toviral reverse transcriptases and proteases. Examples of viruses thatencode these enzymes include the retroviruses (eg. HIV-1, both enzymes,see Turner B. G. and Summers M. F. (1999)), the picornaviruses (eg.,Hepatitis A virus, Wang Q. M. (1999)), and Hepatitis C virus (Kwong A.D. et al. (1999)). Early clinical success observed with anti-HIVlreverse transciptase and protease inhibitors (reviewed by Shafer R. W.and Vuitton D. A. (1999)) has been tempered by the development ofresistance, largely due to mutations in the virally-enoded enzymes(Catucci, M. et al. (1999); Mahalingam, B. et al. (1999); and Palmer, S.et al. (1999)). Highly drug-resistant HIV-1 clinical isolates arecross-resistant to many anti-retroviral compounds in current clinicaldevelopment. Hooker et al. (1996). In these cases of resistance, theviral enzymes retain their catalytic activity because the mutatedversion of the enzyme retains the structure of the wild-type active siteof the enzyme. The compounds of this invention are specifically designedto interact with the active site and be converted by this interactioninto a toxin. Accordingly, the drug resistant viral infections aresensitive to the compounds of this invention that require the activatingenzyme to generate toxin in the infected cell. NB1011 1 is an example ofsuch a compound, directed against TS expressed by mammalian and humancells as well as pathogens.

[0167] Co-Administration

[0168] Co-administration of these compounds with other agents mayprovide unexpected synergistic therapeutic benefit. In theco-administration methods, the compounds are also useful in reducingdeleterious side-effects of known therapies and therapeutic agents, aswell as yet to be discovered therapies and therapeutic agents. In oneaspect, the compounds are combined with a nucleoside transportinhibitor. Suitable nucleoside transport inhibitors include one or moreselected from the group consisting of dipyridamole (DP),p-nitrobenzylthioinosine (NBMPR), 6-benzylaminopurine,2′,3′-dideoxyguanosine, 8-bromoadenine,9-[(2-hydroxyethoxy)methyl]guanine (Acyclovir),9-[(1,3-dihydroxy-2-propoxy) methyl]guanine (Ganciclovir), adenine,hypoxanthine, allopurinol, dilazep, cytochalasin B, lidoflaxine,mioflazine, phloretin, phloridzine, and benzylisoquinoline alkaloids.

[0169] Suitable benzylisoquinoline alkaloids are selected from the groupconsisting of papaverine, ethaverine, laudanosine, noscarpine, andberberine. Additional operative combinations include, but are notlimited to agents or drugs that neutralize or prevent the production oftumor necrosis factor-α (TNF-α) such as an anti-TNF-α antibody orsoluble TNF-α receptor that treat or ameliorate the symptoms associatedwith autoimmune diseases.

[0170] Other examples include, but are not limited to corticosteriods,non-steroidal anti-inflammatory drugs (N-SAIDS), and anti-rheumaticdrugs.

[0171] The use of operative combinations is contemplated to providetherapeutic combinations that may lower total dosage of each componentthan may be required when each individual therapeutic method, compoundor drug is used alone. A reduction in adverse effects may also be noted.Thus, the present invention also includes methods involvingco-administration of the compounds described herein with one or moreadditional active agents or methods. Indeed, it is a further aspect ofthis invention to provide methods for enhancing other therapies and/orpharmaceutical compositions by co-administering a compound of thisinvention. In co-administration procedures, the agents may beadministered concurrently or sequentially. In one embodiment, thecompounds described herein are administered prior to the other activeagent(s), therapy or therapies. The pharmaceutical formulations andmodes of administration may be any of those described herein or known tothose of skill in the art.

[0172] In another aspect, the invention provides a method to enhance thecytotoxity of a compound of this invention against a pathological tissueor cell, containing contacting the cell or tissue with an effectiveamount of a nucleoside inhibitor compound. It further provides a methodsto inhibit the growth of a pathological tissue or, e.g., a cellhyperproliferative cell by contacting the cell with an effective amountof a composition comprising an one or more compounds of this invention.

[0173] Reversing Resistance

[0174] Resistance to chemotherapeutics can be reversed by contacting theresistant cell with an effective amount of a compound of this invention,in vitro or in vivo. Subsequent to successful treatment, the prior (ororiginal chemotherapeutic) can be re-utilized.

[0175] Use of Compounds for Preparing Medicaments

[0176] The compounds of the present invention are also useful in thepreparation of medicaments to treat a variety of pathologies, e.g.,infectious diseases, cancers, autoimmune diseases or inflammatoryconditions. The methods and techniques for preparing medicaments of acompound are known in the art. For the purpose of illustration only,pharmaceutical formulations and routes of delivery are detailed below.

[0177] Thus, one of skill in the art would readily appreciate that anyone or more of the compounds described more fully below, including themany specific embodiments, can be used by applying standardpharmaceutical manufacturing procedures to prepare medicaments to treatthe many disorders described herein. Such medicaments can be deliveredto the subject by using delivery methods known in the pharmaceuticalarts.

[0178] Pharmaceutical Delivery

[0179] Various delivery systems are known and can be used to administera compound or an agent of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, receptor-mediated endocytosisand the like. Methods of delivery include but are not limited to,intra-arterial, intramuscular, intravenous, intranasal, and oral routes.In a specific embodiment, it may be desirable to administer thepharmaceutical compositions locally to the area in need of treatment;this may be achieved by, for example and not by way of limitation, localinfusion during surgery, by injection, or by means of a catheter. Todetermine patients that can be beneficially treated, a tissue sample canbe removed from the patient and the cells are assayed for sensitivity tothe agent.

[0180] Therapeutic amounts can be empirically determined and will varywith the pathology being treated, the subject being treated and theefficacy and toxicity of the compound as well as whether the compound isused alone or in combination with other agents of therapeutic methods.When delivered to an animal, the method is useful to further confirmefficacy of the agent. One example of an animal model is MLR/MpJ-lpr/lpr(“MLR-lpr”) (available from Jackson Laboratories, Bal Harbor, Me.).MLR-lpr mice develop systemic autoimmune disease.

[0181] Administration in vivo can be effected in one dose, continuouslyor intermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration areknown to those of skill in the art and will vary with the compositionused for therapy, the purpose of the therapy, the target cell beingtreated, and the subject being treated. Single or multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician.

[0182] Suitable dosage formulations and methods of administering theagents can be readily determined by those of skill in the art. Forexample, the compounds are administered at about 0.01 mg/kg to about 200mg/kg, alternatively at about 0.1 mg/kg to about 100 mg/kg, oralternatively at about 0.5 mg/kg to about 50 mg/kg. When the compoundsdescribed herein are co-administered with another agent (e.g., assensitizing agents) or therapy, the effective amount may be less thanwhen the agent is used alone.

[0183] The pharmaceutical compositions can be administered orally,intranasally, parenterally or by inhalation therapy, and may take theform of tablets, lozenges, granules, capsules, pills, ampoules,suppositories or aerosol form. They may also take the form ofsuspensions, solutions and emulsions of the active ingredient in aqueousor nonaqueous diluents, syrups, granulates or powders. In addition to anagent of the present invention, the pharmaceutical compositions can alsocontain other pharmaceutically active compounds or a plurality ofcompounds of the invention.

[0184] More particularly, an agent of the present invention alsoreferred to herein as the active ingredient, may be administered fortherapy by any suitable route including oral, rectal, nasal, topical(including transdermal, aerosol, buccal and sublingual), vaginal,parenteral (including subcutaneous, intramuscular, intravenous andintradermal) and pulmonary. It will also be appreciated that thepreferred route will vary with the condition and age of the recipient,and the disease being treated.

[0185] Ideally, the agent should be administered to achieve peakconcentrations of the active compound at sites of disease. This may beachieved, for example, by the intravenous injection of the agent,optionally in saline, or orally administered, for example, as a tablet,capsule or syrup containing the active ingredient.

[0186] Desirable blood levels of the agent may be maintained by acontinuous infusion to provide a therapeutic amount of the activeingredient within disease tissue.

[0187] Screening Assays

[0188] This invention also provides screening assays to identifytherapeutic potentisl of known and new compounds and combinations.

[0189] In one aspect, the assay requires contacting a first samplecomprising suitable cells or tissue (“control sample”) with an effectiveamount of a compound of this invention and contacting a second sample ofthe suitable cells or tissue (“test sample”) with the agent to beassayed. In a further aspect, the test agent is contacted with a thirdsample of cells or tissue comprising normal counterpart cells or tissueto the control and test samples and selecting agents that treat thesecond sample of cells or tissue but does not adversely effect the thirdsample. For the purpose of the assays described herein, a suitable cellor tissue is one involved in hyperproliferative disorders such ascancer, infectious disease, autoimmune disease or a chronic inflammatorycondition. Examples of such include, but are not limited to cell ortissue infected with an infectious agent, cells or tissue obtained bybiopsy, blood, breast cells, colon cells, liver cells, synovial fluid, achondrocyte or an immune cell, such as a T cell, a macrophage, and an NKcell.

[0190] In a further aspect, the cells are tissue are characterized bythe loss of a native tumor suppressor function. In a yet further aspect,the cells or tissue express a target enzyme or overexpress an endogenousintracellular enzyme. In a still further aspect, the cells or tissuehave developed resistance to a drug such as 5-FU.

[0191] For example, the compound or agent to be tested can be activatedby an endogenous intracellular enzyme that is overexpressed ordifferentially expressed in a pathological cell as compared to itsnormal counterpart. An example of such an enzyme includes, but is notlimited to thymidylate synthase. Alternatively, a cell geneticallymodified to differentially express the enzyme or enzymes (containing theappropriate species of enzyme) can be used. Transfection of host cellswith polynucleotides encoding the enzyme can be either transient orpermanent using procedures well known in the art and described by Chen,L. et al. (1996), Hudziak, R. M. et al. (1988), or Carter, P. et al.(1992), and in the experimental section below. The cells can beprokaryotic (bacterial such as E. coli) or eukaryotic. The cells can bemammalian or non-mammalian cells, e.g., mouse cells, rat cells, humancells, fungi (e.g., yeast) or parasites (e.g., Pneumocystis orLeishmania) which cause disease.

[0192] Suitable vectors for insertion of the CDNA are commerciallyavailable from Stratagene, La Jolla, Calif. and other vendors. Theamount of expression can be regulated by the number of copies of theexpression cassette introduced into the cell or by varying promoterusage. The level of expression of enzyme in each transfected cell linecan be monitored by immunoblot and enzyme assay in cell lysates, usingmonoclonal or polyclonal antibody previously raised against the enzymefor immunodetection. (Chen, L. et al. (1996)). Enzymatic assays todetect the amount of expressed enzyme also can be performed as reviewedby Carreras, C. W. and Santi, D. V. (1995) or the method described inthe experimental section below.

[0193] In a further aspect, more than one species of enzyme is used toseparately transduce separate host cells, so that the effect of thecandidate drug with an enzyme can be simultaneously compared to itseffect on another enzyme or a corresponding enzyme from another species.

[0194] The compounds and/or compositions can be directly added to thecell culture media and the target cell or the culture media is thenassayed for the amount of label released from the candidate compound ifthe compound contains a detectable label. Alternatively, cellular uptakemay be enhanced by packaging the compound into liposomes using themethod described in Lasic, D. D. (1996) or combined with cytofectins asdescribed in Lewis, J. G. et al. (1996).

[0195] In yet a further aspect, the assay requires at least two celltypes, the first being a suitable control cell. The second cell type isof the same type or tissue as the control cell but differs in thatpathogenesis toward disease has begun. In one aspect, pathogenesis isdetermined enzymatically by noting enhanced or over expression of anendogenous intracellular enzyme that activates the compound into a toxicentity. Amplification of genes associated with drug resistance can bedetected and monitored by a modified polymerase chain reaction (PCR) asdescribed in Kashini-Sabet, et al. (1988), Houze, T. A. et al. (1997),U.S. Pat. No. 5,085,983, or the method described herein. Acquired drugresistance can be monitored by the detection of cytogeneticabnormalities, such as homogeneous chromosome staining regions anddouble minute chromosomes both of which are associated with geneamplification. Alternative assays include direct or indirect enzymeactivity assays, each of which are associated with gene amplification(e.g., Carreras, C. W. and Santi, D. V. (1995)) and other methodologies(e.g. polymerase chain reaction or immunohistochemistry (Johnson, P. G.et al. (1997)). These methods also provides the means to detectsubclinical evidency of therapy or a therapeutic effect.

[0196] The assays are useful to predict whether a subject will besuitably treated by this invention by delivering a compound orcomposition to a sample containing the cell to be treated and assayingfor treatment which will vary with the pathology. In one aspect, thecell or tissue is obtained from the subject or patient by biopsy.Applicants provide kits for determining whether a pathological cell or apatient will be suitably treated by this therapy by providing at leastone composition of this invention and instructions for use.

[0197] This invention further provides a method for screening forcompounds that are selectively converted to a toxin by an activatingenzyme by providing cells that express an activating enzyme andcontacting the cells with a candidate compound. At least one test cellexpresses the pathogen's version of the enzyme (wild-type or mutated)and another test cell is a cell sample from the host organism which may,or may not express its own version of the enzyme. One then assays forconversion of the compound into toxic agents by the activating enzymeproduced by the pathogen. As used herein, the test cells can beprokaryotic or eukaryotic cells infected with the pathogen oralternatively, transformed to express the activating enzyme. Forexample, a prokaryotic E. coli which does not endogenously express theactivating enzyme TS is a suitable host cell or target cell.Alternatively, the test cell can be an infected cell isolated from thesubject, or a cultured cell infected with the pathogen. The cell canhave a control counterpart (lacking the target enzyme), or in a separateembodiment, a counterpart genetically modified to differentially expressthe target enzyme, or enzymes (containing the appropriate species oftarget enzyme). More than one species of enzyme can be used toseparately transduce separate host cells, so that the effect of thecandidate drug on a target enzyme can be simultaneously compared to itseffect on another enzyme or a corresponding enzyme from another species.

[0198] In another embodiment, a third target cell is used as a positivecontrol because it receives an effective amount of a compound, such as,for example, the compounds shown below, which have been shown to bepotent compounds.

[0199] In another embodiment, transformed cell lines, such asras-transformed NIH 3T3 cells (ATCC, 10801 University Blvd., Manassas,Va., 20110-2209, U.S.A.) are engineered to express variable andincreasing quantities of the target enzyme of interest from cloned cDNAcoding for the enzyme. Transfection is either transient or permanentusing procedures well known in the art and described in Sambrook, etal., supra. Suitable vectors for insertion of the cDNA are commerciallyavailable from Stratagene, La Jolla, Calif. and other vendors. The levelof expression of enzyme in each transfected cell line can be monitoredby immunoblot and enzyme assay in cell lysates, using monoclonal orpolyclonal antibody previously raised against the enzyme forimmuno-detection. The amount of expression can be regulated by thenumber of copies of the expression cassette introduced into the cell orby varying promoter usage. Enzymatic assays to detect the amount ofexpressed enzyme also can be performed as reviewed by Carreras and Santi(1995), supra, or the methods described below.

[0200] The test cells can be grown in small multi-well plates and isused to detect the biological activity of test compounds. For thepurposes of this invention, the successful candidate drug will block thegrowth or kill the pathogen but leave the control cell type unharmed.

[0201] The candidate compound can be directly added to the cell culturemedia or previously conjugated to a ligand specific to a cell surfacereceptor and then added to the media. Methods of conjugation for cellspecific delivery are well known in the art, see e.g., U.S. Pat. Nos.5,459,127; 5,264,618; and published patent specification WO 91/17424(published Nov. 14, 1991). The leaving group of the candidate compoundcan be detectably labeled, e.g., with tritium. The target cell or theculture media is then assayed for the amount of label released from thecandidate compound. Alternatively, cellular uptake may be enhanced bypackaging the compound into liposomes using the method described inLasic, D. D. (1996) or combined with cytofectins as described in Lewis,J. G. et al. (1996).

[0202] Compounds, agents and combinations thereof, identified by thismethod are further provided herein.

[0203] In one embodiment, the assay of the effect of the compound isprovided by analysis of intracellular metabolites of the compound. Inthis embodiment, the compound contains a detectable label that ismonitored during conversion of the compound to toxic agent by theactivating enzyme. In an alternative embodiment, the candidate compoundis detectably labeled, e.g., e.g., fluorescent marker, or aradioisotope. In a further aspect, the detectable label comprises atleast two or more variable isotopes of the same atom, e.g., bromine. Inthis embodiment, one can assay for the modification of the compound intotoxic byproducts by mass spectrometry of the reaction products. Onemeans to accomplish this assay is by use of mass spectrometry asdescribed in more detail below.

[0204] Using the above screen, one also can pre-screen several compoundsagainst samples taken from a subject such as a human patient. One canuse the screen to determine the most effective compound and therapy foreach pathology or pathogen and subject.

[0205] Kits

[0206] Applicants also provide kits for determining whether apathological cell, tissue or patient will be suitably treated by thistherapy. Additionally, kits for performance of the assays are provided.These kits contain at least one compound or composition of thisinvention and instructions for use.

[0207] The following examples are intended to illustrate, but not limit,the invention.

MATERIALS AND METHODS General Synthesis Procedures

[0208] Synthesis of Nueleoside Compounds

[0209] Synthesis of 5-substituted pyrimidine derivatives can beaccomplished by methods known in the art, for example as described inApplicant's patent literature, PCT/US98/16607 and PCT/US99/01332.

[0210] One method requires treatment of 5-chloromercuri-2′-deoxyuridinewith haloalkyl compounds, haloacetates or haloalkenes in the presence ofLi₂PdCl₄ to form, through an organopalladium intermediate, the 5-alkyl,5-acetyl or 5-alkene derivative, respectively (Wataya, Y. et al. (1979)and Bergstrom, D. E. et al. (1984)). Another example of C5-modificationof pyrimidine nucleosides and nucleotides is the formation ofC5-trans-styryl derivatives by treatment of unprotected nucleotide withmercuric acetate followed by addition of styrene or ring-substitutedstyrenes in the presence of Li₂PdCl₄ (Bigge, et al. (1980)).

[0211] Pyrimidine deoxyribonucleoside triphosphates can be derivatizedwith mercury at the 5 position of the pyrimidine ring by treatment withmercuric acetate in acetate buffer at 50° for 3 hours (Dale, et al.(1973)). Such treatment also would be expected to be effective formodification of monophosphates. Alternatively, a modified triphosphatecan be converted enzymatically to a modified monophosphate, for example,by controlled treatment with alkaline phosphatase followed bypurification of monophosphate. Other moieties, organic or nonorganic,with molecular properties similar to mercury but with preferredpharmacological properties could be substituted. For general methods forsynthesis of substituted pyrimidines see, for example, U.S. Pat. Nos.4,247,544, 4,267,171, and 4,948,882 and Bergstrom, D. E. et al. (1981).The above methods would also be applicable to the synthesis ofderivatives of 5-substituted pyrimidine nucleosides and nucleotidescontaining sugars other than ribose or 2′-deoxyribose, for example2′-3′-dideoxyribose, arabinose, furanose, lyxose, pentose, hexose,heptose, and pyranose. An example of a 5-position substituent is thehalovinyl group, e.g. (E)-5-(2-bromovinyl)-2′-deoxyuridylate (Barr, P.J. et al. (1983)).

[0212] Alternatively, 5-bromodeoxyuridine, 5-iododeoxyuridine, and theirmonophosphate derivatives are available commercially from Glen Research,Sterling, Va. (USA), Sigma-Aldrich Corporation, St. Louis, Mo. (USA),Moravek Biochemicals, Inc., Brea, Calif. (USA), ICN, Costa Mesa, Calif.(USA) and New England Nuclear, Boston, Mass. (USA).Commercially-available 5-bromodeoxyuridine and 5-iododeoxyuridine can beconverted to their monophosphates either chemically or enzymatically,through the action of a kinase enzyme using commercial availablereagents from Glen Research, Sterling, Va. (USA) and ICN, Costa Mesa,Calif. (USA). These halogen derivatives could be combined with othersubstituents to create novel and more potent antimetabolites.

[0213] In one aspect, the structures at the 5-position of the compound,analogs and derivatives thereof are referred to as the tethers becausethey connect a proposed leaving group (toxophore) to the heterocycle.

[0214] In another aspect, the tether also contains a spacer between thetoxin and the pyrimidine ring can be unsaturated, e.g., vinyl, allyl,and propargyl units are simple, small, and readily accessiblesynthetically. The vinyl and allyl units have the advantage that theycan be prepared in either of two non-interconvertible geometric isomericforms. Alternatively, synthesis based on the structure of BVdUmonophosphate and features a proposed leaving group/toxin directlyattached to the terminus of a (poly)vinyl substituent at C5 of thepyrimidine ring. This is the vinyl tether approach. A yet furtherapproach is based on the structure of TFPe-dUMP and is similar to thevinyl tether approach but has a methylene unit separating the proposedleaving group/toxin and the unsaturated unit and thus contains an allylor propargyl unit. This is the allyl tether approach.

[0215] 5-Alkylidenated 5,6-dihydrouracils similar in structure to theintermediate common to both the vinyl and allyl tether approachmechanisms have been synthesized recently (Anglada, J. M. et al. 1996).A C5 methylene intermediate produced by the enzyme thymidylate synthaseTS was demonstrated by trapping studies (Barrett, J. E. et al. (1998)).

[0216] The compounds of Formula B are defined by the structure of theuracil base, or modified uracil base present. These classes arecompounds where: 1) the base is a furano-pyrimidinone derivative ofuracil; 2) the base is 6-fluoro uracil; 3) the base is 4-hydrazonesubstituted uracil derivative; and 4) the base is uracil. In one aspect,the uracil or modified uracil derived base is used to synthesizecompounds substituted with toxic leaving groups at the 5 position,attached by an electron conduit tether at this 5 position, and includingan appropriate spacer moiety between the electron conduit and the toxicleaving group. The compounds can be unphosphorylated, 5′ monophosphate,5′ phosphodiester, or 5′ protected (“masked”) deoxyuridines orcomparable derivatives of alternative carbohydrate moieties, asdescribed below. Protected 5-substituted deoxyuridine monophosphatederivatives are those in which the phosphate moiety has been blockedthrough the attachment of suitable chemical protecting groups. Inanother embodiment, 5-substituted uracil or uridine derivatives areadministered to cells containing nucleoside kinase activity, wherein the5-substituted uracil/uridine derivative is converted to a 5-substituteduridine monophosphate derivative. Uridine derivatives may also bemodified to increase their solubility, cell penetration, and/or abilityto cross the blood-brain barrier.

[0217] Synthesis of Compounds with Propargyl Tethers

[0218] The synthesis of propargylic and allylic alcohol-equipped2′-deoxyuridines are reported in the literature. For example, Barr, P.J. and Robins, M. J. (1981) and Balzarini, J. et al. (1985).

[0219] Both 5-mercuri- (Ruth, J. L. et al. (1978)) and 5-iodouridines(Robins, M. J. et al. (1981)) readily condense with alkenes and alkynesin the presence of a palladium catalyst to afford C5 tether-equippeduridines. The latter route is the more often employed (Robins, M. J. etal. (1982) and Asakura, S. et al. (1988) and (1990)). High-yieldingcondensations of protected 5-iodo-2′-deoxyuridines witht-butyidimethylsilyl propargyl ether (Graham, D. et al. (1998); DeClercq, E. et al. (1983), methyl propargyl ether (Tolstikov, V. V. etal. (1997)) and even propargyl alcohol itself (Chaudhuri, N. C. et al.(1995) and Goodwin, J. T. et al. (1993)) have been achieved. The3-hydroxy-1-propynyl substituent introduced by the latter reaction canalso be accessed by DIBAL-H reduction of a methacrylate group (Cho, Y.M. et al. (1994)), itself arising from the same Heck reaction used inthe synthesis of BVdU. These palladium-catalyzed reactions can be usedto condense very long and elaborately-functionalized propargyl-basedtethers to 5-iodo-2′-deoxyuridines. (Livak, K. J. et al. (1992) andHobbs, F. W. Jr. (1989)). (Z)-Allyl-based tethers are generated by thepartial hydrogenation of a propargylic precursor over Undiar catalyst(Robins, M. J. et al. (1983)) whereas the (E)-allyl-based ones are bestprepared by Heck coupling of an (E)-tributylstannylated ethylene (Crisp,G. T. (1989)).

[0220] Closely following the literature procedures, at-butyldimethylsilyl propargyl ether-equipped 3′, 5′-di-O-protected2′-deoxyuridine (Graham, D. et al. (1998), and De Clercq, E. et al.(1983)) can be prepared and a portion of it, converted to thecorresponding (Z)-allyl ether, (Robins, M. J. et al. (1983)) is reduced.Because the TBAF-mediated removal of a TBDMS group generates an oxyanionthat can be functionalized in situ, these TBDMS-protected propargyl- and(Z)-allytic-tethered nucleosides can serve as convenient precursors tosome of the toxophore-equipped targets. For the (E)-allyl alcoholequipped nucleoside, the known O-tetrahydropyranyl ether derivative isprepared by the literature Heck coupling of an (E)-tributylstannylatedethylene (Crisp, G. T. (1989)).

[0221] Using a two step literature protocol (Phelps, M. E. et al. (1980)and Hsiao, L. Y. et al. (1981)), the propargylic and (E) and (Z)-allylicalcohols are converted to their corresponding bis-aziridinylphosphoramidates or thiophosphoramidates.

[0222] Synthesis of Furano-Pyrimidinones

[0223] Synthesis of furano-pyrimidinones begins with synthesis of a C5propargylic—alcohol-equipped 2′-deoxyuridine. Furano-pyrimidinonecompounds are then be formed from the O-tetrahydropyranyl etherderivative described above. Synthesis proceeds by reaction of the secondcarbon of the propargyl bond with the oxygen attached to the C4 positionof the pyrimidine ring to yield a fluorescent furano-pyrimidinone whichcan be readily separated from the reaction mix. Such compounds providean additional basis for synthesis of compounds through variouscombinations of specific electron conduits, spacers and toxic leavinggroups.

[0224] Furo[2,3-d]pyrimidinone nucleosides (represented by the abovegeneric structure) were prepared by condensing 2′,3′-di-O-p-toluoyl or2′,3′-di-O-acetyl-5-iodo-2′-deoxyuridine with1-(tetrahydropyranyloxy)-2-propyne (Jones, R. G. and Mann, M. J. (1953))under conditions known to promote the formation of these fluorescentcompounds (Robins, M. J. et al.(1983)). Base-catalyzed removal of thecarbohydrate protecting groups gave the6-(tetrahydropyran-2-yloxymethyl)-substituted bicyclic nucleoside whichwas either subjected to standard acidic THP group hydrolysis (TFA inCH₂Cl₂) or was regioselectively 5′-phosphoramidated by the sameprocedure used to prepare BVdU-PA and 5FUdR-PA. After thephosphoramidation, the THP group can be removed by acidic hydrolysis.

[0225] Compounds Based on Furano-Pyrimidinones

[0226] Examples of synthesis of compounds having a structure of theclass shown are as follows.

[0227] Proposed toxic R⁴ leaving groups can be attached to the furan-2methyl alcohol using methods similar to those employed to attach toxicleaving groups to the hydroxyl on the C5 propargyl uridine compound, asexplained above. A variety of alternative toxic leaving groups areenvisioned. In addition, modifications to the length and composition ofthe R² electron conduit component and of the composition of the R³spacer element are also envisioned.

[0228] Compounds based on furano-pyrimidinones can also consist ofvariously modified phosphoramidates. A method for synthesis of suchphosphoramidate compounds is accomplished by reacting a 2-deoxy3′-hydroxy, 5′-hydroxy unprotected nucleotide with a phosphochloridatein the presence of an HCI scavenger. In one aspect, thephosphochloridate comprises a phosphorus substituent which is derivedfrom an amino acid such as alanine. For example, the phosphochloridatecan be phenyl-L-methoxyalanine phosphorochloridate.

[0229] C6 Fluoro Uridine and C4 Hydrazone Based Compounds

[0230] The introduction of fluorine at the C6 position can besynthesized by following the synthetic descriptions of Krajewskas andShugar (1982), who describe the synthesis of a number of 6 substituteduracil and uridine analogs.

[0231] Chemistry facilitating substitutions at the C4 position of thepyrimidine base are known by those skilled in the art. Examples ofliterature descriptions include Wallis, et al. (1999); Negishi, et al.(1996), Barbato, et al. (1991), Barbato, et al. (1989) and Holy et al.(1999). These synthetic techniques also enable combinations ofsubstitutions, for instance at the C4 and C5 positions of the pyrimidinering (Pluta, et al. (1999)) or the C2 and C4 positions of the pyrimidinering (Zeid, et al. (1999)).

[0232] In another embodiment of the invention, compounds are synthesizedby addition of alternative electron conduits, spacer moieties and toxicleaving groups to either the C6 fluoro-uridine base or the C4 hydrazonemodified pyrimidine. Methods described above for synthesis of2-deoxyuridine based compounds can again be employed for the synthesisof such molecules.

[0233] Synthesis of Nucleoside Phenyl Methoxyalaninyl Phosphoramidates

[0234] The use of phosphoramidates as phosphate prodrugs for nucleotideswas reported by McGuigan, C. et al. (1993) and McGuigan, C. et al.(1994). The phospharamidates were synthesized by reacting2′,3′-dideoxynucleosides with phenyl methoxyalaninyl phosphorochloridate(PMPC).

[0235] Since only one hydroxyl group is present, these reactions usuallyproceed smoothly. In compounds where more than one hydroxyl group ispresent, the appropriately protected nucleoside may be required. Sincethe 5′-OH group of 2′-deoxynucleosides is much less hindered than the3′-OH group, selective phosphoramidation with PMPC is possible undercarefully controlled conditions. Both BVdU and 5FUdR condensed with PMPCin the presence of N-methylimidazole in anhydrous CH₂Cl₂ to give thecorresponding phosphoramidates. In both cases, the desired product wasreadily separable from the starting material using column chromatographyon silica gel. The synthetic scheme is summarized below.

EXAMPLES 1 AND 2 Synthesis of Compounds with Propargyl Tethers

[0236] Using the general synthetic procedure described supra,bis-aziridin-1-yl-phosphinic acid 3-[2-deoxyuridin-5-yl]-prop-2-ynylester was synthesized and analyzed by ¹H NMR to yield the followingresult: ¹H NMR ((CD₃)₂SO). Salient features: δ8.28 (d, 1, H6), 6.10(pseudo-t, 1, H1′), 5.26 (m, exchanges with D₂O, 1, 3′-OH), 5.13 (m,exchanges with D₂O, 1, 5′-OH), 4.81 (q or dd, 2, propargyl-CH₂), 4.24(m, 1, H3′), 3.57 (m, 2, 5′-CH₂), 2.15-2.0 (m, 8, aziridine-CH₂).

[0237] Bis-aziridin-1-yl-phosphinothioic acid3-[2-deoxyuridin-5-yl]-prop-2-ynyl ester was also synthesized andanalyzed by ¹H NMR to yield the following result: ¹H NMR ((CD₃)₂SO).Salient features: δ8.29 (d, 1, H6), 6.10 (pseudo-t, 1, H1′), 5.22 (m,exchanges with D₂O, 1, 3′-OH), 5.10 (m, exchanges with D₂O, 1, 5′-OH),4.88 (q or dd, 2, propargyl-CH₂), 4.31 (m, 1, H3′), 3.52 (m, 2, 5′-CH₂),2.15-2.0 (m, 8, aziridine-CH₂).

EXAMPLES 3 TO 8 Synthesis of Furano-pyrimidinones

[0238] Using the general synthetic procedure described supra, thefollowing compounds were prepared.

Example 3

[0239]3-(2-Deoxy-β-D-ribofuranosyl)-6-(tetrahydropyran-2-yloxymethyl)furo[2,3-d]pyrimidin-2(3H)-one.¹H NMR ((CD₃)₂SO) δ8.80 (s, 1, H4), 6.74 (s, 1, H5), 6.16 (pseudo-t, 1,H1′), 5.27 (d, exchanges with D₂O, 1, 3′-OH), 5.12 (t, exchanges withD₂O, 1, 5′-OH), 4.72 (m, 1, THP-H2), 4.56 (q, 2, CH₂OTHP), 3.92 (m, 1,H4′), 3.64 (m, 2, 5′-CH₂), 2.40 (m, 1, H2′a), 2.03 (m, 1, H2′b), 1.68and 1.50 (m, 8, THP). Low-resolution mass spectrum (DCI—NH₃) on bis-TMSderivative, m/z 323 (B+TMS+H⁺), 511 (MH⁺), 583 (M+TMS⁺).

Example 4

[0240]3-(2-Deoxy-β-D-ribofuranosyl)-6-(hydroxymethyl)furo[2,3-d]pyrimidin-2(3H)-one.¹H NMR ((CD₃)₂SO) δ12.0 (bs, 1, OH), 8.24 (s, 1, H4), 6.53 (s, 1, H5),5.51 (pseudo-t, 1, H1′), 4.42 (m, 2, CH₂OH). Low-resolution massspectrum (DCI—NH₃), m/z 167 (B+2H⁺), 184 (B+NH₄ ⁺).

Example 5

[0241]1-[6-(Tetrahydropyran-2-yloxymethyl)furo[2,3-d]pyrimidin-2(3H)-on-3-yl]-2-deoxy-β-D-ribofuranos-5-yl phenylmethoxy-L-alaninylphosphoramidate. ¹HNMR ((CD3)2SO) complicated due topresence of diastereomers. Salient features: δ8.62 and 8.59 (each s,each 1, H4), 7.4-7.1 (m, 5, PhO), 6.61 and 6.60 (each s, each 1, H5),6.25 (m, 1, H1′), 4.56 (q, 2, propargyl-CH₂), 3.56 and 3.54 (each s,each 3, CO₂Me), 2.0 (m, 1, H2′b), 1.22 (m, 3, alaninyl-α-Me).Low-resolution mass spectrum (DCI—NH3), m/z 167 (B+2H⁺), 184 (B+H⁺+NH₄⁺-THP).

Example 6

[0242]1-[6-(Hydroxymethyl)furo[2,3-d]pyrimidin-2(3H)-on-3-yl]-2-deoxy-β-D-ribofuranos-5-ylphenyl methoxy-L-alaninylphosphoramidate. ¹H NMR (CDCl₃) complicated dueto presence of diastereomers. Salient features: δ8.5 (s, 1, H4), 7.4-7.1(m, 5, PhO), 6.36 and 6.30 (each s, each 1, H5), 6.23 (m, 1, H1′), 3.67and 3.65 (each s, each 3, CO₂Me), 2.69 (m, 1, H2′a), 2.10 (m, 1, H2′b),1.35 (m, 3, alaninyl-α-Me). Low-resolution mass spectrum (DCI—NH₃), m/z525 (MH⁺), 595 (MNH₄ ⁺).

Example 7

[0243] The 4-nitrophenyl ether derivative of5-(3-hydroxy-1-propynyl)-2′-deoxyuridine was prepared according tostandard ether synthesis as shown below.

Example 8 5-[3-(4-Nitrophenoxy)-1-propynyl]-2′-deoxyuridine

[0244] A solution of pre-dried 5-(3-hydroxy-1 -propynyl)-2′-deoxyuridine(Robins, M. J. et al. (1983)) (565 mg, 2 mmol) in 40 mL of anhydrous THFunder argon was treated with 4-nitrophenol (696 mg, 5 mmol),triphenylphosphine (787 mg, 3 mmol), and diisopropyl azodicarboxylate(590 liters, 3 mmol), and the reaction mixture heated at 60° C. untilthe solution was clear, and then 1 hour longer. The mixture was allowedto cool to 23° C. and then it was evaporated onto SiO₂ and purified bychromatography using MeOH/CH₂Cl₂ as eluent to afford 107 mg (13%) of thedesired ether product: melting point 112-118° C. H NMR ((CD₃)₂SO) δ11.65(s, exchanges with D₂O, 1, NH), 8.29 (s, 1, H6), 8.24 (d, J=9.3 Hz, 2,m-ArH), 7.23 (d, J=9.3 Hz, 2, o-ArH), 6.09 (pseudo-t, 1, H1′), 5.17 (s,2, propargyl-CH₂), 4.22 (m, 1, H3′), 3.80 (m, 1, H4′), 3.59 (m, 2,5′-CH₂), 2.13 (pseudo-t, 2, 2′-CH₂). Low-resolution mass spectrum(DCI—NH₃) onper-trimethylsilyated material, m/z 547 [M(TMS)₂H⁺], 565[M(TMS)₂NH₄ ⁺], 620 [M(TMS)₃H⁺].

EXAMPLE 9 5-(4-Carbethoxy-1,3-butadienyl)-2′-dexoyuridine

[0245] (a)5-(Carbomethoxyvinyl)-2′-deoxyuridine-3′,5′-bis(tetrahydro-2H-pyran-2-yl)ether(I)

[0246] A slurry of 5-(carbomethoxyvinyl)-2′-deoxyuridine (3.0 g, 9.6mmol), 3,4-dihydro-2H-pyran (22 mL, 21.3 mmol) and pyridiniump-toluenesulfonate (PPTS, 0.242 g, 0.96 mmol) in dimethylformamide (DMF,5 mL) was stirred at 50° C. for 18 hours. The resulting solution wasconcentrated in vacuo (bath temperature 45° C.) to give a thick, paleyellow oil. The oil was dissolved in EtOAc and the solid was filtered.The solution was again concentrated. The oil obtained was purified bycolumn chromatography on silica gel using 50-75% EtOAc/hexane as eluentto give 3.81 g (85%) of pure product as a colorless oil.

[0247] (b) 5-(3-Hydroxyprop-1-enyl)-2′-deoxyuridine-3′,5′-bis(tetrahydro-2H-pyran-2-yl)ether (II)

[0248] A solution of (I) (3.5 g, 7.27 mmol) in CH₂Cl₂ (14 mL) was cooledto −78° C. in a dry ice/acetone bath. Diisobutylaluminum hydride(DIBAL-H) in toluene (1.0M, 24 mL, 24.0 mmol) was added dropwise over 2hours while the temperature was maintained at −78° C. The solution wasstirred at −78° C. for an additional 2 hours and MeOH (2.5 mL) was addeddropwise to destroy any excess DIBAL-H. The reaction mixture wascannulated into a mixture of 30% citric acid solution (50 mL), ice (25g) and EtOAc (30 mL) over ca. 20 minutes. The phases were separated andthe aqueous phase was extracted with EtOAc (2×25 mL). The combinedorganic phase was washed with saturated NaHCO₃ (20 mL) and brine (20mL), dried over MgSO₄ and concentrated to give 3.288 g (100%) ofcolorless oil

[0249] (c)5-(3-Oxoprop-1-enyl)-2′-dexoyuridine-3′,5′-bis(tetrahydro-2H-pyran-2-yl)ether(III)

[0250] To a solution of crude (II) obtained from above (1.988 g, 4.4mmol) in CH₂Cl₂ (9 mL) was added solid pyridinium dichromate (PDC; 1.82g, 4.8 mmol) with water cooling. The suspension was stirred while aceticacid (0.4 mL) was added dropwise. The water bath was removed and thereaction was stirred at room temperature for 1 hour. The crude productwas filtered through a pad of florisil (2×2.5 cm) and the florisilwashed with 35 mL EtOAc. The brown solution obtained was filteredthrough another column of florisil (3.5 cm diam×2.5 cm height). Thefiltrate was concentrated to give 1.273 g (64% yield) of very lightbrown oil.

[0251] (d)5-(4-Carbethoxy-1,3-butadienyl)-2′-dexoyuridine-3′,5′-bis(tetrahydro-2H-pyran-2-yl)ether(IV)

[0252] (Carbethoxymethylene)triphenylphosphorane (0.32 mg, 0.92 mmol)was added to a solution of the crude aldehyde (III) (0.344 g, 0.77mmol). The solution darkened and turned rust color. After 1 hour, (III)was completely consumed as judged by thin layer chromatography. Thesolvent was evaporated and the crude product was purified by columnchromatography on silica gel using 35-45% EtOAc/hexane as eluent. Thepure product (0.310 g, 78% yield) was obtained as colorless oil.

[0253] (e) 5-(4-Carbethoxy-1,3-butadienyl)-2′-dexoyuridine (V)

[0254] 5-(4-Carbethoxy-1,3-butadienyl)-2′-dexoyuridine-3′,5′-bis(tetrahydro-2H-pyran-2-yl)ether (IV) (0.637 g,1.22 mmol) was dissolved in MeOH (1.5 mL) and PPTS (0.049 g, 0.16 mmol)was added. The solution was stirred at 50° C. for 7.5 hours and left atroom temperature overnight. A white precipitate was formed. The reactionmixture was cooled to 0° C. and filtered to give pure (V) as a whitesolid (0.188 g). The filtrate was concentrated and chromatographed onsilica gel using 50-100% EtOAc/hexane as eluent to give a further 0.180g product. The total yield of the product was 0.368 g (86%).

[0255]¹H NMR (DMSO-d₆): 1.22 (3H, t, J=7 Hz), 2.17 (2H, br t, J=5.5 Hz),3.55-3.75 (2H, m), 3.81 (1H, m), 4.12 (2H, q, J=7 Hz), 4.25-4.28 (1H,m), 5.19 (1H, t, J=4.8 Hz), 5.27 (1H, d, J=4.1 Hz), 5.98 (1H, d, J=14.5Hz), 6.14 (1H, t, J=6.3 Hz), 6.75 (1H, d, J=14.5 Hz), 7.18-7.30 (2H, m),8.30 (1H, s), 11.56 (1H, s).

EXAMPLE 10 5-(4-Carbomethoxy-1,3-butadienyl)-2′-dexoyuridine (Va)

[0256] A solution of triethylamine (3.9 mL, 28.2 mmol) in dioxane (12mL) was deareated by bubbling nitrogen through for 15 minutes. Palladiumacetate (0.60 g, 0.26 mmol) and triphenylphosphine (0.183 g, 0.70 mmol)were added and the solution was heated at 70° C. for 20 minutes to givea dark brown solution. 5-Iodo-3′-deoxyuridine (5.0 g, 14.1 mmol) andmethyl 2,4-pentadienoate (2.5 g, 22.3 mmol) were added and the mixturewas heated under reflux for 15 hours. The solvent and volatilecomponents were evaporated in vacuo and the residue was partitionedbetween water (15 mL) and EtOAc (15 mL). The phases were separated andthe aqueous phase was extracted twice with EtOAc (10 mL each). Thecombined organic phase was washed with brine and concentrated. Theresidue was dissolved in MeOH (15 mL) and allowed to cool to roomtemperature. The solid formed was collected by filtration, washed with asmall quantity of MeOH and dried in vacuo to give 0.38 g brown powder.

[0257]¹H NMR (DMSO-d₆): 2.17 (2H, t, J=6.4 Hz), 3.55-3.70 (2H, m), 3.66(3H, s), 3.82 (1H, q, J=3.6 Hz), 4.27 (1H, m), 5.18 (1H, t, J=4.9 Hz),5.26 (1H, d, J=4.5 Hz), 5.99 (1H, d, J=14.4 Hz), 6.14 (1H, d, J=6.4 Hz),6.74 (1H, d, J=14.8 Hz), 7.20-7.35 (2H, m), 8.30 (1H, s), 11.56 (1H, s).

[0258] The filtrate from above was concentrated and chromatographed onsilica gel using 60-100% EtOAc/hexanes as eluent to give another 0.70 gof product as a brown foam. The combined yield was 1.08 g (22.6%).

EXAMPLE 11 5-(4-Carboxy-1,3-butadienyl)-2′-dexoyuridine (VI) Method I

[0259] 5-(4-Carbethoxy-1,3-butadienyl)-2′-dexoyuridine (V, from Example9) (0.449 g, 1.28 mmol) was dissolved in 2N NaOH (3 mL) and stirred at25° C. After 20 minutes, a precipitate was formed and TLC showed thatthe starting material was completely consumed. The mixture was cooled to0° C. and acidified to pH 1 with 2N HCl. The resulting off-white solidwas filtered off, washed with water and dried in vacuo to give 0.225 g(54%) product.

[0260]¹H NMR (DMSO-d₆): 2.12-2.19 (2H, m), 3.50-3.70 (2H, m), 3.75-3.85(1H, m), 4.24-4.29 (1H, m), 5.19 (1H, t, J=4.8 Hz), 5.27 (1H, d, J=4.2Hz), 5.80-5.95 (1H, m), 6.14 (1H, t, J=6.4 Hz), 6.60-6.75 (1H, m),7.15-7.25 (2H, m), 8.26 (1H, s), 11.56 (1H, s), 12.16 (1H, br s).

[0261] The filtrate and washings were combined and evaporated todryness. The resulting sticky yellow solid was dissolved in MeOH fromwhich a white precipitate was formed. The solid was filtered off to givean additional 0.200 g of product.

Method II

[0262] The title compound can also be prepared from5-(4-carbomethoxy-1,3-butadienyl)-2′-dexoyuridine (Va, from Example 10)in comparable yield as mentioned above.

EXAMPLE 12 5-(4-Bromo-1E,3E-butadienyl)-2′-dexoyuridine (VIIa) and5-(4-Bromo-1E,3Z-butadienyl)-2′-dexoyuridine (VIIb)

[0263] To a solution of 5-(4-carboxy-1,3-butadienyl)-2′-dexoyuridine(VI) (0.200 g, 0.62 mmol) in DMF (1 mL) was added KHCO₃ (0.185 g, 1.84mmol) and the mixture was stirred for 20 minutes at 25° C. A solution ofN-bromosuccinimide (0.117 g, 0.65 mmol) in DMF (0.3 mL) was addeddropwise. Smooth gas evolution (CO₂) occurred throughout the addition.The resulting brown suspension was stirred for 2 hours at 25° C. atwhich time TLC showed that (VI) was completely consumed. Water (10 mL)was added to the suspension and the resulting solution was extractedwith EtOAc (2×15 mL). The extract was dried over MgSO₄ and the solventwas evaporated in vacuo to give a yellow solid (178 mg, 80% yield)consisting of a mixture of two isomers as shown by ¹H NMR. The crudeproduct was separated by semi-preparative HPLC (reversed phase C18column) using 20% acetonitrile in water as the mobile phase to give thefollowing isomers:

[0264] 5-(4-Bromo-1E,3Z-butadienyl)-2′-dexoyuridine: retention time 10.5minutes; ¹H NMR: (DMSO-d₆): 2.11-2.18 (2H, m), 3.50-3.70 (2H, m), 3.80(1H, distorted q, J=3.5 Hz), 4.25 (1H, br s), 5.08 (1H, br s), 5.25 (1H,br s), 6.15 (1H, t, J=6.5 Hz), 6.40 (1H, d, J=7 Hz), 6.53 (1H, d, J=15.6Hz), 6.83 (1H, dd, J=7, 10 Hz), 7.39 (1H, dd, J =10, 15.6 Hz).

[0265] 5-(4-Bromo-1E,3E-butadienyl)-2′-dexoyuridine: retention time 15.1minutes; ¹H NMR (DMSO-d₆): 2.12-2.16 (2H, m), 3.50-3.70 (2H, m), 3.80(1H, q, J=3.2 Hz), 4.26 (1H, m), 5.13 (1H, br s), 5.25 (1H, br s), 6.14(1H, t, J=6.5 Hz), 6.36 (1H, d, J=15.6 Hz), 6.67 (1H, d, J=13.1 Hz),6.84(1H, dd, J=11, 13.1 Hz), 7.04 (1H, dd, J=11, 15.6 Hz).

EXAMPLE 13

[0266] Using the procedures mentioned in Example 11, Method II, thefollowing compounds can be obtained in a similar fashion:5-(4-chloro-1,3-butadienyl)-2′-dexoyuridine (using N-chlorosuccinimidein place of N-bromosuccinimide in Step B);5-(4-iodo-1,3-butadienyl)-2′-dexoyuridine (using iodine in sodiumidodide in place of N-bromosuccinimide).

EXAMPLE 14 Phenyl N-methoxy-L-alaninyl Phosphorochloridate

[0267] L-alanine methyl ester hydrochloride (245.8 g; 1.76 mol) wasplaced in a 12 liter three-neck round bottom flask (equipped with amechanical stirrer and thermometer) followed by 4.0 liters ofdichloromethane. The mixture was stirred for 15 minutes at roomtemperature. Phenyl phosphodichloridate (370.0 g; 1.76 mol) was added tothe mixture and stirring was continued for 15 minutes at roomtemperature. The flask was placed in the bath with dry ice and thestirring was continued for 20 minutes until a uniform suspension wasformed.

[0268] Freshly distilled tri-n-butylamine (626.5 g; 3.38 mol) was addeddropwise (˜90 minutes) with vigorous stirring to the reaction mixture sothat the temperature inside the flask was held at ˜0° C. The bath wasremoved and the stirring was continued for 6 hours at room temperature.The solution was concentrated to ˜2.84 liters by evaporating severalportions of the mixture on a rotary evaporator and the mixture wassealed under argon and stored at −20° C. The product was 85% pure byphosphorus NMR to give an estimated concentration ofphenylmethoxyalaninyl phosphochloridate of ˜0.5M.

EXAMPLE 15 5-(2-Bromovinyl)-2′-deoxyuridine phenyl N-methoxy-L-alaninylphosphoramidate (NB1011)

[0269] The reaction was performed under argon atmosphere.5-(2-bromovinyl)-2′-deoxyuridine (BVdU) (204 g; 612 mmol) was placed inthree-neck 3 liter round bottom flask equipped with mechanical stirrer.The flask was placed in ice-water bath and 1600 mL (˜800 mmol) ofphenylmethoxyalaninyl phosphochloridate reagent were added using anaddition funnel over 15 minutes with vigorous stirring of the reactionmixture, followed by the addition of 100 mL of N-methylimidazole over 5minutes using syringe. After 5 minutes the mixture became clear andafter 10 minutes the ice-water bath was removed to allow the mixture towarm up to room temperature while stirring was continued. The reactionwas monitored by reversed phase HPLC and was complete in 3 hours. Thereaction was quenched by the addition of 100 mL of methanol and themixture was evaporated to an oil, re-dissolved in 6 liters ofdichloromethane and passed through 800 g of silica gel. The majorportion of BVdU-PA, referred to herein as NB1011, was passed through thecolumn during the loading and finally the elution of NB1011 wascompleted by passing 5 liters of 5% methanol in dichloromethane. Allfractions containing NB1011 were combined and evaporated to an oil, theresidue was dissolved in 4 liters of ethyl acetate and the mixture wasextracted with water (2×2 liters). The organic layer was dried withsodium sulfate, filtered, and washed with ethyl acetate (3×300 mL). Thecombined filtrate and washings were evaporated to produce a lightlycolored white foam; total weight ˜540 g.

[0270] The crude product was purified by two silica gel chromatographyusing 0-5% MeOH in CH₂Cl₂ and 10% MeOH in CH₂Cl₂, respectively, aseluent. The yield of product (>98% pure) was 64 g.

EXAMPLE 16

[0271] Using the methods described in Example 15, the phenylN-methoxy-L-alanyl phosphoramidates of the following nucleosides wereprepared:

[0272] 5-(4,4-dibromo-1,3-butadienyl)-2′-deoxyuridine;

[0273] 5-(2-chlorovinyl)-2′-deoxyuridine;

[0274] 5-trifluoromethyl-2′-deoxyuridine;

[0275] 5-(4-carbethoxy-1,3-butadienyl)-2′-deoxyuridine;

[0276] 5-(4-carbomethoxy-1,3-butadienyl)-2′-dexoyuridine;

[0277] 5-(4-bromo-1E,3E-butadienyl)-2′-deoxyuridine;

[0278] 5-(4-bromo-1E,3Z-butadienyl)-2′-deoxyuridine;

[0279] 5-(trimethylsilylethynyl)-2′-deoxyuridine;

[0280] 5-(ethynyl)-2′-deoxyuridine;

[0281] 5-(1-decynyl)-2′-deoxyuridine;

[0282]3-(2′-deoxy-β-D-ribofuranosyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one;and

[0283]3-(2′-deoxy-β-D-ribofuranosyl)-6-octyl-2,3-dihydrofuro[2,3-d]pyrimidin-2-one.

EXAMPLE 17

[0284] Using the methods described in Examples 14 and 15, the followingamino acid phosphoramidate derivatives of5-(2-bromovinyl)-2′-deoxyuridine were prepared:

[0285] Phenyl (benzoxy-L-alaninyl) phosphoramidate;

[0286] Phenyl (methylene cyclopropoxy-L-alaninyl) phosphoramidate;

[0287] Phenyl (cyclohexoxy-L-alaninyl) phosphoramidate;

[0288] Phenyl (iso-propoxy-L-alaninyl) phosphoramidate;

[0289] Phenyl (methylene tert-butoxy-L-alaninyl) phosphoramidate;

[0290] Phenyl (cycloheptoxy-L-alaninyl) phosphoramidate;

[0291] Phenyl (cyclooctoxy-L-alaninyl) phosphoramidate;

[0292] Phenyl (methylene adamantoxy-L-alaninyl) phosphoramidate; and

[0293] Phenyl (methoxy-L-tryptophanyl) phosphoramidate.

[0294] Chemical assays for products, for example, where a reactionproduct is an anti-metabolite of the bromovinyl-derivatives of dUMP, aredescribed in the Examples provided below or by Barr, P. J. et al.(1983).

EXAMPLE 18 Cell and Enzyme Based Assays

[0295] Expression of thymidylate synthase in human normal tissues. TheTS expression level in normal human tissues was examined in order toestimate the systemic toxicity of the compound(s) activated bythymidylate synthase. The relative TS mRNA levels in brain, heart,kidney, spleen, liver, colon lung, small intestine, stomach muscle,testis, ovary, uterus, prostate, thyroid gland, salivary gland, adrenalgland, skin, PBL and bone marrow tissues were determined by usingRT-PCR. It has been shown that TS mRNA levels in most of these tissueswere equal to or less than that in colon tissue, except that in bonemarrow (1.25 fold), ovary (1.38 fold) and testis tissues (2.13 fold).However, the average TS mRNA level in colon cancer samples was 4.6 foldmore than that in their matched normal colon tissue samples. This resultsuggests that compounds which are activated by overexpressed TS in coloncancer would have no or little toxicity to normal human tissues.

[0296] Transcript levels of human thymidylate synthase in multiplenormal tissues were investigated by PCR amplification. Panel of cDNAs ofhuman tissues were obtained from OriGene Technologics, Inc. (Rockville,Md.). PCR reactions were perfromed in a volume of 25 μl, containing cDNA(100×), 3 mM MgCl_(2,), 50 mM KCl 20 mM Tris-Cl, pH 8.4, 0.2 mM of eachdNTP, 0.2 μM of thymidylate synthase sense and antisense primers and1.25 units of Taq DNA polymerase (obtained from Promega, Madison, Wis.).The reaction mixtures were incubated at 94° C. for 2 min, followed by 12cycles of 40 sec incubations at 94° C., 1 min incubation at 58° C., andthen 1 min incubation at 72° C.25 μl reaction buffer contained 0.2 μMβ-actin primers, 0.2 μM of thymidylate synthase primers, 3 mM MgCl₂, 50mM KCl, 20 mM Tris-cl, pH 8.4, 0.2 mM of each dNTP and 1.25 units of TaqDNA polymerase were added to achieve a final concentration of 0.2 μM ofthymidylate synthase primers and 0.1 μM β-actin primers, bringing thereaction volume to 50 μl. PCR reaction was continued to a total of 36cycles, followed by a 7 min incubation at 72° C.

[0297] 10 μL of PCR products were resolved by electrophoresis in a 2%agarose gel, followed by staining with SYBR Gold nucleie acid gel stain(obtained from Molecular probes, Eugene, Oreg.). The DNA bandscorresponding to thymidylate synthase were quantified and normalized tothat of β-actin by Molecular Dynamics Storm. The quantified expressionlevels were expressed as values relative to that of colon.

[0298] RT-PCR analysis of matched normal and tumor tissues. Transcriptlevels of human thymidylate synthase in colon cancer tissues and matchednormal colon tissues were quantified by using Reverse RT-PCRamplification. Oligonucleotide primers for amplification of the humanthymidylate synthase and β-actin were designed as following: thymidylatesynthase sense primer 5′-GGGCAGATCCAACACATCC-3′ (SEQ ID NO. 1)(corresponding to bases 208-226 of thymidylate synthase cDNA sequence,Genbank Accession No. X02308), antisense primer5′-GGTCAACTCCCTGTCCTGAA-3′ (SEQ ID NO. 2) (corresponding to bases564-583), β-actin sense primer 5′-GCCAACACAGTGCTGTCTG-3′ (SEQ ID NO. 3)(corresponding to bases 2643-2661 of β-actin gene sequence, GenbankAccession No. M10277) and antisense primer 5′-CTCCTGCTTGCTGATCCAC-3′(SEQ ID NO. 4) (corresponding to bases 2937-2955).

[0299] Human colon tumor tissues and matched normal tissues wereobtained from Cooperative Human Tissue Network (CHTN, Western Division,Cleveland, Ohio). Total RNAs were isolated using Tri pure isolationreagent (obtained from Boehringer Mannheim Corp., Indianapolis, Ind.),followed manufactureis protocol. To monitor for possible DNAcontamination, the primers for amplification of β-actin were designed tospan the exon4/intron5/exon5 junction. Genomic DNA template leads to a313 bp β-actin fragment, and cDNA template generates a 210 bp product.

[0300] Reverse transcriptions were performed, using SuperScriptpreamplification system (Gibco/BRL, Gaithersburg, Md.). 3 μg total RNAwas applied in a volume of 20 μl buffer to conduct reverse transcriptionreaction, followed manufacture's protocol.

[0301] PCR reactions were performed in a volume of 96 μl, containing 5μl of cDNA mixture from reverse transcription reaction, 3 mM MgCl₂, 50mM KCl, 20 mM Tris-Cl, pH 8.4, 0.2 mM of each dNTP, 0.3 μM ofthymidylate synthase sense and antisense primers and 5 units of Tag DNApolymerase (obtained from Promega, Madison, Wis.). The reaction mixtureswere incubated at 94° C. for 3 min, followed by 9 cycles of 1 minincubation at 94° C., 1 min incubation at 58° C., and then 1 minincubation at 72° C. After 9 cycles, human β-actin primers in 4 μl wereadded to achieve a final concentration of 0.2 μM, bringing the finalreaction volume to 100 μl. PCR reaction was continued to a total of 30,32 or 34 cycles, followed by a 7 min incubation at 72° C.

[0302] 10 82 L of PCR products were resolved by electrophoresis in 2%agarose gel, followed by staining with SYBR Gold nucleic acid gel stain(obtained from Molecular probes, Eugene, Oreg.). Result ofquantification indicated that amplification of thymidylate synthase andβ-actin was linear between cycles 30 and 34. The DNA bands correspondingto thymidylate synthase were quantified and normalized to that of actinby Molecular Dynamics Storm. The quantified expression levels wereexpressed as values of ratio between TS and β-actin.

[0303] Cell lines and transfection. HT1080 cells were grown in PRM11640medium supplemented with 10% fetal calf serum, and transfected withGFP-TS expression vector. 48 hours after, transfection cells weretripsinized and replated in culture medium containing 750 μg/ml G418.After selection with G418 for two weeks, surviving cells were sortedbased upon fluorescence expression. One clone with higher fluorescenceexpression (named as TSH/HT1080) and one clone with lower fluorescenceexpression (named as TSL/HT1080) were selected and expanded into cellslines. The stable HT1080 cells transfected with pEGFP-C3 were used ascontrol.

[0304] Construction of GFP-TS expression vector. A cDNA fragmentencoding conserved region of human thymidylate synthase (amino acids 23to 313) was obtained by PCR amplification using following primers: Senseprimer, 5′-CGGAAGCTTGAGCCGCGTCCGCCGCA-3′ (SEQ ID NO. 6) and antisenseprimer, 5′-GAAGGTACCCTAAACAGCCATTTCCA-3′ (SEQ ID NO. 7). The cDNA wascloned into HindIII and KpnI sites of mammalian expression vectorpEGFP-C3 ( Clontech Laboratories. Inc., Palo Alto, Calif.), in-framewith GFP sequence. The cDNA insert was confirmed by DNA sequencing.

[0305] Western blot analysis. Human normal and cancer cells were grownin RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells weregrown till confluent in 100 mm culture dish and lysed in 0.5 ml of RIPAbuffer ( 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 0.1%SDS, 0.5% Deoxycholic acid, sodium salt and protease inhibitors ).Protein concentrations were determined by using BCA-200 protein assaykit (obtained from Pierce, Rockford, Ill.). 15 μg of total protein fromeach cell strain/line was resolved by 12% SDS-PAGE. The separatedproteins were transferred onto PVDF membrane, followed by immunoblotwith human thymidylate synthase monoclonal primary antibody(manufactured by NeoMarkers, Fremont, Calif.) and horseradish peroxidaselinked sheep anti-mouse Ig secondary antibody (obtained from Amersham,England). The ECL plus kit (Amersham) was used for detection ofimmunoreactivity. The bands corresponding to thymidylate synthase werequantified and normalized to that of tubulin by Molecular DynamicsStorm. The quantified expression levels were expressed as valuesrelative to that of cell strain CCD 18co.

[0306] TS Activity Assay by Tritium Release from dUMP-³H. Cells wereplated in 24 well plates to a density of 30,000 cells/plate andincubated for 16 hours to allow adhesion to the plastic surface of theplate.

[0307] Immediately prior to the thymidylate synthase assay, the mediawas replaced with RPMI+10% dialyzed fetal calf serum. 0.5 μCi of5-[³H]deoxyuridine was added to each well, and plates were incubated for60 minutes at 37° C. without additional CO₂. [³H] release was measuredby adsorbing 5-[³H]deoxyuridine to activated charcoal (10% in 1×PBS) for5 minutes at room temperature. After centrifugation for 5 minutes at13,000 RPM, the amount of [³H] in the supernatant was determined byliquid scintillation counting.

[0308] Growth Inhibition Studies. Cells growing exponentially weretransferred to 384-well flat bottom tissue culture plates. All celltypes were plated at a density of 500 cells per well in 25 μL ofcomplete medium (RPMI 1640+10% fetal bovineserum+antibiotics/antimycotics). After 24 hours (day 0), 25 μL ofcomplete medium containing the experimental compounds over the doserange of 10^(ñ3) to 10^(ñ10) M were added in triplicate. Drug exposuretime was 120 hours (day 5), after which growth inhibition was assayed. 5μL of the redox indicator, alamarBlue, was added to each well (10% v/v).After 4 hours incubation at 37° C., fluorescence was monitored at 535 nmexcitation and 595 nm emission.

[0309] Concentration vs. relative fluorescence units (RFU) were plotted,and sigmoid curves were fit using the Hill equation. IC₅₀, indicated bythe inflection point of the curve, is the concentration at which growthis inhibited by 50%.

[0310] Enzyme Preparation. Cloned human thymidylate synthase plasmidpBCHTS was subcloned into E. coli. BL21 (DE3)/pET-28a(+) (Novagen) usingthe Ndel ñSacI insertion site, in order to add an amino terminal Histag. Enzyme was expressed in E. coli. by induction with IPTG, andpurified by affinity chromatography on a Ni²⁺ His Bind metal chelationresin (Novagen). The column Ni²⁺ His Bind metal chelation column waswashed with 20 mM Tris pH 7.9, 5 mM imidazole, 0.5M NaCl; thymidylatesynthase activity was eluted with 20 mM Tris pH 7.9, 60 mM imidazole,0.5M NaCl.

[0311] Enzyme Assays and Kinetic Measurements. Thymidylate synthaseassays were done in 96 well Costar UV transparent plates in a reactionvolume of 200 μL, consisting of 40 mM Tris pH 7.5, 25 mM MgCl₂, 1 mMEDTA, 25 mM-mercaptoethanol, 125M dUMP, and 65 μM N5, N10-methylenetetrahydrofolate indicated. Tetrahydrofolate stock solutions wereprepared by dissolving tetrahydrofolic acid (Sigma) directly into 0.2MTris pH 7.5, 0.5 M-mercaptoethanol; stock solutions were stored at −80°C. N5, N10-methylene tetrahydrofolate was prepared by adding 12 μl of3.8% formaldehyde to 1 ml of a 0.65 mM solution of tetrahydrofolate andincubating for 5 minutes at 37° C. N5, N10-methylene tetrahydrofolatewas kept on ice and used within 2 hours of preparation.

[0312] Conversion of BVdUMP to fluorescent product(s) by thymidylatesynthase was measured in 200 μl thymidylate synthase reactionscontaining 125M BVdUMP in 96 well Dynex Microfluor Black “U” bottommicrotiter plates using an excitation wavelength of 340 nm and emissionwavelength of 595 nm. Fluorescence was measured with a TecanSpectrafluor Plus fluorimeter.

[0313] Enzyme kinetic constants (K_(m) and V_(max)) were determined forthe human thymidylate synthase substrates dUMP and BVdUMP using theenzyme assay conditions described above. Results are shown in Table 3.The initial rates of the enzyme reactions was determined by measuringthe increase in A₃₄₀ for the reaction with dUMP, and decrease in A₂₉₄for the reaction with BVdUMP. The catalytic efficiency of the enzyme(K_(cat)/K_(m)) was calculated from the kinetic constants K_(m) andV_(max).

[0314] Liquid Chromatography/Mass Spectroscopy. Cells were washed threetimes with PBS at room temperature, then subjected to freeze/thaw lysisin 5 ml PBS. Cell extracts were centrifuged for 10 minutes at 10KRPM,then adsorbed to Sep-Pak C₁₈ and washed with 10 ml PBS. BVdUMP waseluted with 1 ml distilled water. LC/MS samples were analyzed by reversephase chromatography on a C₁₈ column using a linear gradient of 0.1%formic acid-0.1% formic acid/95% acetonitrile. Mass spectroscopy wasdone with a Micromass Quattro II triple quadropole spectrometer.

[0315] Tomudex Inhibition of NB1011 Cytotoxicity. MCF7-TDX weretransferred to a 384 well assay plate at 500 cells in 25 μL completemedium per well. After 24 hours (day 0), 25 μL complete mediumcontaining a combination of NB1011 in doubling serial dilutions from 1mM and tomudex at discrete concentrations (0,1,10,100,1000 nM) wereadded in duplicate. Drug exposure time was 120 hours (day 5) after whichgrowth inhibition was measured with alamarBlue as described above inGrowth Inhibition Studies (above).

[0316] Reversal of Resistance. The origin and characteristics of thehuman breast cancer MCF7 TDX cell line have been previously described(Drake, et al. (1996)). Briefly, MCF-7 breast cancer cells were selectedin vitro for resistance to Tomudex by continuous exposure to stepwiseincreases in TDX concentrations up to 2.0 μM. A resistant subline wasselected for resistance to NB1011 by continuous exposure of the parentalMCF7 TDX cell line to medium supplemented without TDX but with 50 μMNB1011, a concentration approximately 16 times higher than the IC₅₀ forNB1011 in the parental MCF7 TDX cell line. After a dramatic initial cellkilling effect, resistant colonies emerged, and vigorously growingmonolayers were formed. TS protein level and IC₅₀ for 5-FU, TDX, andNB1011 were determined for the resultant MCF7 TDX/1011 cell line asdescribed in above by western blot and the alamarBlue cytotoxicityassay, respectively.

[0317] Analysis of NB1011 in TS-expressing, 5-FU resistant, H630-10colon carcinoma xenografts in vivo. H630-10 colon cancer cells, selectedfor resistance to 5-FU in vitro, express high levels of thymidylatesynthase, and form xenografts in athymic mice. Following cell expansionex-vivo H630-10 were injected subcutaneously (S.Q.) at 1.5×10⁷cells/tumor in the mid-back region of 4-6 week old, female, CD-1(nu/nu), athymic mice (Charles River Laboratories, Wilmington, Mass.).Tumor volumes, calculated as the product of length, width, and depth,were monitored twice weekly by serial micrometer measurements by asingle observer. Six animals were randomly assigned to each treatmentgroup and statistical tests were performed (single-factor ANOVA) toassure uniformity in starting tumor volumes between treatment andcontrol groups at the beginning of the experiment. NB1011 wasadministered by intraperitoneal (I.P.) or intratumoral (IT) injection.The dosage of experimental agents tested were as follows: Group 1: DMSOvehicle control solution (IP), Group 2: 5-FU (15 mg/kg×5 days IP=the MTDfor 5-FU in this model), Group 3: NB1011=1.25 mg×5 days (IP), Group 4:NB1011=2.5 mg×5 days (IP), Group 5: NB1011=3.5 mg×5 days (IP), Group 6:DMSO control (IT), Group 7: NB1011=1.25 mg×5 days (IT), and Group 8:NB1011=2.5 mg×5 days (IT). These doses were based on independentdose-finding experiments conducted in our laboratory and were near themaximum-tolerated dose of NB1011 for this specific age and strain offemale athymic mice. To assure accurate dosing, drug doses wereindividualized based upon animal weights determined immediately prior toeach injection. Treatment with control solution or NB1011 was initiated10 days status post xenograft inoculation at which time xenograftvolumes measured 45-68 mm³. Differences in day 25 xenograft volumesbetween groups were analyzed by single-factor ANOVA of the logtransformed tumor volume data. Experimental athymic mice were maintainedunder aseptic conditions in a dedicated room in the UCLA Animal CareFacility. The University of California, Los Angeles has an AnimalWelfare Assurance document on file with the Office of Protection fromResearch Risks, Laboratory Animal Safety Assurance Number A-3196-01. Allexperiments were closely supervised by the UCLA veterinarian. Euthanasiatechniques employed by UCLA are supported by the Panel of the AmericanVeterinary Medical Association. The University of California, LosAngeles experimental animal program and facilities are accredited by theAmerican Association for the Accreditation of Laboratory Animal Care.The personnel performing the animal procedures/manipulations describedin this protocol are technically competent in those procedures and havereceived their training on the use of animals in research, as requiredby the Animal Welfare Act of 1985.

In vitro Reaction of BVdUMP with Human Thymidylate Synthase

[0318] 1. The Cell-free Processing of BVdUMP by rHuTS GeneratesFluorescent Product(s)

[0319] The cell-free processing of BVdUMP by L. casei TS has been shownto create potentially reactive intermediates (Barr, et al. (1983)). Forthis reason it has been thought that processing of BVdUMP by TS leads toirreversible inactivation of human TS (Balzarini (1987)). The cell-basedexperiments by DeClercq, Balzarini and colleagues (Balzarini (1987);Balzarini (1993); and Balzarini (1995)) support the concept that, onceBVDU is converted to the monophosphate in cells (via herpes virusthymidine kinase), then it binds to and inactivates the Hu TS enzymeduring processing. However, the actual reaction of human TS with BVdUMPhas never been adequately characterized. Santi and colleagues (Barr, P.J. et al. (1983)) utilized a bacterial TS for their work to showgeneration of product from the BVdUMP+TS reaction, and DeClercq andcolleagues utilized cells and cell lysates, not purified human TS(Balzarini (1987)); Balzarini, (1993); and Balzarini (1995)).

[0320] The interaction of BVdUMP with purified recombinant human TS(rHuTS) was revisited. When BVdUMP was incubated with rHuTS in thestandard reaction mixture, the reaction results in the formation offluorescent product(s) (FIG. 1). The time dependent increase influorescence is accompanied by a decrease in the initial BVdUMPconcentration. The product(s) produced have been partiallycharacterized, and appear to be exocyclic pyrimidine nucleotidederivatives.

[0321] This result is surprising because previous results supported theidea that TS reaction with BVdUMP should inactivate the human TS enzyme(Balzarini, et al. (1987), (1993), and (1995)). Because the reactionsdescribed above were done in a cell-free system with purifiedcomponents, it remained possible that the intracellular milieu couldprovide components that would result in TS inactivation followingconversion of NB1011 to the free nucleotide monophosphate inside thecell. This issue is addressed in more detail below.

[0322] 2. Comparative Reaction Kinetics of dUMP and BVdUMP with rHuTS

[0323] Previously reported work by Barr, et al.(1983), utilizing the L.casei TS (Balzarini (1987), (1993), (1995) and (1995b)) using cells andcell lysates, leaves unclear whether the reaction of BVdUMP with humanTS will result in irreversible inactivation of the enzyme. To addressthis question, the kinetics of interaction of BVdUMP with rHuTS, in thepresence or absence of dUMP, were determined.

[0324] Competitive inhibition is most consistent with a reaction inwhich BVdUMP does not inactivate the TS enzyme. To help further clarifythis situation, an extended incubation of rHuTS with BVdUMP was done inorder to measure any inactivation that may occur over a period of timelonger than that in which the kinetics were performed (FIG. 2).

[0325] These data show that even after a 20 hour incubation of rHuTSwith BVdUMP, little or no enzymatic inactivation is apparent as measuredby rate of conversion of THF DHP dUMP as substrate. This result isconsistent with the hope for ability of overexpressed TS to convertBVdUMP into cytotoxic metabolites in cells, preferentially in cellswhich overexpress TS, and finally, without inactivating the enzyme.

[0326] 3. Characterization of BVdUMP reaction with TS: Cofactors andInhibitors

[0327] The best characterized reaction of TS is the conversion of dUMPto dTMP. This reaction involves the transfer of a methylene group fromN5,N10-methylene tetrahydrofolate (THF) to the C-5 position of dUMP(Carreras, C. W. and Santi, D. V. (1995)). This reaction is dependentupon the cofactor (THF), and is inhibited by the uridylate mimic,5F-dUMP, which, upon reaction with the enzyme, results in the formationof a stable complex and loss of enzymatic activity. A second wellcharacterized inhibitor of TS activity is Tomudex, which occupies thefolate binding site of the TS homodimer, prevents the binding of THF,and blocks TS activity in the cell (Drake (1996) and Touroutoglou andPazdur (1996)). As part of a preliminary effort to characterize thereaction of rHuTS with BVdUMP, the effects of 5F-dUMP, Tomudex andcofactor were compared on the reaction of the enzyme with dUMP andBVdUMP. These experiments (Table 3) show that, similarly to the case ofdUMP, 5F-dUMP can prevent conversion of BVdUMP to fluorescentproduct(s). In addition, Tomudex can also prevent product formation fromboth dUMP and BVdUMP. However, consistent with earlier reported resultswith L. casei TS (Barr, et al. (1983)), THF is not required for theconversion of BVdUMP to fluorescent product(s). In addition, the datashown in Table 3 also demonstrate that THF stimulates the production offluorescent product(s) in the BVdUMP reaction with rHuTS. This result isnot expected from the earlier data reporting that THF has no effect onthis reaction (Barr, P. J. et al. (1983)), and illustrates a potentiallyimportant possibility that cofactors, or cofactor agonists, likeleucovorin, could modulate the reaction of BVdUMP with human TS.

[0328] Analysis of the Michaelis-Menton kinetics of this reaction showedthat inhibition of BVdUMP by dUMP fits the expected form for competitiveinhibition, consistent with both nucleotides behaving as substrates forrHuTS.

[0329] Previously reported data with the L. casei TS indicated thatBVdUMP is 385 times less efficient a substrate as dUMP (Barr, P. J. etal. (1983); Santi, D. V. (1980)). The experiments reported herein haveshown that this situation is quite different with the human enzyme. ForrHuTS the relative catalytic efficiency of dUMP compared with BVdUMP is60×. This represents a >6.4 fold increase in catalytic efficiency ascompared to endogenous substrate. The previous result with L. casei TSleads to the prediction that the efficiency of enzymatic reaction withinthe cell would be too low for NB1011 to be an effective therapeuticsubstrate, since it would have to compete with large amounts ofendogenous dUMP. The discovery reported herein, ie. that the humanenzyme has a >6.4-fold improved efficiency of conversion of BVdUMP, isan important factor enabling utility of NB1011. The increased efficiencyof BVdUMP utilization by the human enzyme as compared to the L. caseienzyme also establishes that species specific substrates are possibleand can be designed. The ability to specifically inhibit heterologousenzymes via binding to species specific regions on the surface of L.casei vs. human TS has recently been reported (Stout (1999) and Costi,et al. (1999)). Differences in specificity relating to the active siteof TS, which is composed of the most highly conserved regions of theprotein (Carreras, C. W. and Santi, D. V. (1995)) is surprising and hasnot been reported previously.

[0330] Products of the cell-free enzymatic reaction of rHuTS with BVdUMPwere analyzed by mass spectroscopy. The two molecular structures of theproducts have masses that are consistent with the mass of molecular ionsdetected in TS reaction mixtures following incubation of BVdUMP withpurified rHuTS. Knowledge of the products of this reaction may be usedto understand the final mechanism of action of NB1011. In addition, thisinformation could be used to design novel chemotherapeutics, since theproducts of the TS-BVdUMP reaction could, themselves, be potentiallyused as chemotherapeutics.

[0331] 4. NB1011 is Converted to the Monophosphate in Tumor Cells

[0332] NB1011 is converted from the phosphoramidate to the monophosphateform in cells, as a prerequisite for binding to TS. To determine whetherthis crucial step in conversion was taking place advantage was taken ofan unusual property of the bromine atom, i.e. that it exists in naturein two equally abundant isotopic forms. This situation allows detectionof the bromine containing monophosphate by focusing the massspectrometry analysis on the predicted mass ions of BVdUMP (411 and 413daltons). H630 R10 tumor cells (which express high levels of TS) wereincubated with 100 FM NB1011. Extracts of treated cell lysates wereprepared as described herein. Detection using mass spectroscopy,following an initial purification with liquid chromatography relied uponformation of the unprotected nucleotide mass ions of BVdUMP which haveidentical retention times on reverse phase chromatography. Results areshown in FIGS. 3A and 3B.

Characterization of the Cytotoxic Activity of NB1011

[0333] 1. The Tumor/normal Cell Screen

[0334] As an initial step in characterizing the biological activity ofNB1011, a large series of normal and tumor cell types were tested in thealamarBlue assay for sensitivity to both NB1011 and 5-fluorouracil.

[0335] Assays were carried out as described in Methods, above.Therapeutic index is calculated as the ratio of the average IC₅₀ fornormal cells to the average IC₅₀ for tumor cells. All assays were doneat least three times. See Table 5.

[0336] These data show that NB1011 has met the primary design goal forTS ECTA compounds, i.e. increased potency on tumor cells vs. normal celltypes. Overall, NB1011 is about 2-fold more cytotoxic to tumor cells vs.normal cells, while 5-FU is 3-fold more toxic to normal cells than it isto tumor cells. The total benefit of NB1011 is therefore (2)×(3)=6-foldimprovement in therapeutic index for NB1011 as compared with 5-FU. Acritical tactic that allows for selection of chemotheraputics with apositive therapeutic index is screening of activity on both normal andtumor cell types. This approach has not been consistently employed inthe field of new cancer drug discovery. For instance, screening of newcandidate compounds on normal cell types is part of the National CancerInstitute's screening procedure (Curt (1996)).

[0337] 2. NB1011 Does Not Inactivate TS in Vivo

[0338] The results described above indicate that BVdUMP, generatedintracellularly from NB1011, is unlikely to inactivate TS during itstransformation to product(s). However, the cell free system is differentfrom the intracellular milieu. In order to further explore thisquestion, cell-based assays for TS activity were performed. In theseexperiments exogenous 5-(3H) deoxyuridine is added to cell culturemedium and the release of tritiated water is monitored (Carreras, C. W.and Santi, D. V. (1995) and Roberts (1966)). FIG. 4 shows that thepresence of NB1011 in cell culture media reduces the rate at which[³H]₂O is released from 5-[³H]dUMP. In order to determine whether thisis the result of irreversible inhibition of TS, NB1011-treated cellswere allowed to briefly recover in fresh culture media, then assayed forTS activity. Cells that have been allowed to recover in culture medialacking NB1011 have the same level of TS activity as untreated cells.This result supports the proposal that NB1011 does not irreversiblyinactivate the TS enzyme following intracellular processing.

[0339] An additional approach was taken to understanding whether NB1011might interfere with cell growth primarily by inactivating TS. Thisapproach is based upon thymidine rescue of TS-blocked cells. Cells whichare blocked by Tomudex or by 5FdUMP (following treatment by 5FdUrd) donot make dTMP by de novo synthesis. For this reason, they survive onlyby scavenger mechanisms. One of the important scavenger mechanisms isutilization of extracellular thymidine. Thymidine incorporated by targetcells can be converted to dTMP, usually by thymidine kinase, and thuscontinue DNA synthesis. Other pathways for use of exogenous thymidinehave also been described If an important mechanism for NB1011 activityis via inhibition of endogenous TS, then the cytotoxicity should berelieved when thymidine is added to the cell culture media. For thisexperiment, a number of tumor cell lines were screened for theirsensitivity to Tomudex and 5FdUrd, and ability to be rescued from theseagents via thymidine supplementation. The normal colon epthelial cell,CCD18co, was used because of its measurable sensitivity to NB1011, 5FUdRand Tomudex. Experiments were carried out as described by (Patterson, etal. (1998)) with or without 10 μM thymidine, except that the alamarBlueassay was employed to determine cytotoxicity. The results showed a15-fold rescue from Tomudex (IC₅₀ change from 6.5 nM to 95 nM), agreater than 590-fold rescue from 5FudR (from an IC₅₀ of less than 0.01μM to greater than 5.9 μM), and a slight decrease in the absence ofthymidine to 223 μM in the presence of 10 μM thymidine.

[0340] 3. Relationship Between TS Level and NB1011-mediated Cytotoxicityon Tumor Cell Lines

[0341] Confirmation that TS participates in NB1011-mediated cytotoxicitywas established using several approaches: 1). The activity of NB1011 wasexamined on normal colon cells vs. high TS expressing, 5FU-resistant,tumor cells; 2). transfection of TS into a tumor cell background, andgenerating clonal derivatives which differ primarily by TS expressionlevel, but are otherwise very similar; and 3). Use of a specificinhibitor of TS, Tomudex, to decrease intracellular TS activity.

[0342] In the initial analysis, of NB1011 and 5FUdR-mediatedcytotoxicity were compared on the CCD18co normal colon epithelial celltype and H630R^(10, 5)FU-resistant colon tumor cell line (Copur, S. etal. (1995)). This allows a determination of cytotoxicity vs. normalcells (CCD18co) as well as a measure of cytotoxicity vs. drug-resistanttumor cells (H630R10), which overexpress TS. This is important because asignificant limitation to current chemotherapeutics is their toxicity tonormal tissues. The results are presented in Table 5.

[0343] This experiment shows that 5FUdR is about 18-fold more toxic tonormal colon cells (CCD18co) than to 5FU-resistant H630R10 tumor cells.This negative therapeutic index characterizes the major limitation ofcurrent chemotherapy, i.e. its toxicity to normal tissue. Such anegative therapeutic index has also been reported for doxorubicin(Smith, et al. (1985) and Smith, et al. (1990)). In contrast to 5FUdR,however, NB1011 has more than an 11 -fold improved activity ondrug-resistant H630R10 cells (IC₅₀=216.7 μM) vs. normal colon epithelialcells (IC₅₀ greater than 2500 μM). This result suggests that: 1).Activity of NB1011 is more pronounced on high TS expressing tumor cells;and 2). A total improvement in therapeutic index of (18)×(11)=198-foldis achievable using TS ECTA compounds vs. 5FUdR.

[0344] 4. Overexpression of TS in HT1080 Tumor Cells Enhances TheirSensitivity to NB1011

[0345] Activation of NB1011 requires several steps. These include cellpenetration conversion to the nucleotide monophosphate, binding to TS,and subsequent toxic metabolism. The precise mechanisms of cellpenetration and conversion are not fully defined. Cell entry may dependin part on nucleoside transport mechanisms (Cass, et al. (1998)).Similarly, processing from the phosphoramidate to the monophosphateemploys poorly defined mechanisms (Abraham, et al. (1996)).

[0346] These results are particularly significant because theydemonstrate, in a fairly uniform genetic background, that increasing TSlevels predicts enhanced sensitivity to NB1011. In addition, the dataalso show that increasing TS levels predicts resistance tofluoropyrimidines, a result consistent with reports in the literature(Copur, et al. (1995); Banerjee, et al. (1998)).

[0347] 5. Inhibitors of NB1011-mediated Cytotoxicity

[0348] Tomudex is a chemotherapeutic that acts primarily via inhibitionof TS. If NB1011 exerts cytotoxicity via the TS enzyme, then inhibitionof TS with Tomudex should decrease NB1011-mediated cytotoxicity. To testthis hypothesis directly, Tomudex-resistant MCF7 cells, whichoverexpress TS 11-fold compared to the parental MCF7 cell line, wereexposed to NB1011 in the presence of increasing concentrations of TDX.Cells were plated and exposed to indicated concentrations of compound(s)as described above. Results are shown in Table 7.

[0349] The data show that blockade of TS using the specific inhibitorTomudex, results in up to about 25-fold inhibition of NB1011 -mediatedcytotoxicity. These results support the concept that activity of NB1011results from its metabolism by TS.

[0350] To further characterize the intracellular metabolism of NB1011,combination experiments with leucovorin (LV; 5-formyltetrahydrofolate)were performed. This experiment was initiated because we had observedthat THF stimulates production of fluorescent product(s) in thecell-free reaction of BVdUMP and rHuTS. It was hypothesized that if thefluorescent products are related to the cytotoxic effects of NB1011,then enhancing intracellular levels of THF by providing LV in theculture media would also enhance NB1011-mediated cytotoxic effects.Surprisingly, in the presence of 3 μM LV, NB1011 activity on the H630R10cell line was diminished by more than 90%, compared to NB1011 alone, asdetermined in the alamarBlue assay. The fact that NB1011 activity isabolished by LV, which supplements intracellular reduced folate pools,suggests that NB1011 may work in part by diminishing these pools.Alternatively, LV (or a metabolite) could directly impact the metabolismof BVdUMP by interfering with its interaction with TS.

[0351] To explore whether LV could directly impact the reaction ofBVdUMP with TS, reactions were carried out+/−THF with BVdUMP, or withTHF+dUMP, and+/−Methotrexate (MTX), LV or Tomudex (TDX).

[0352] The results (Table 8) are surprising in two respects: 1).Although an increase in fluorescent product was noted from BVdUMP in thepresence of THF, a decreased rate of substrate consumption (BVdUMP)utilization occurs in the presence of the cofactor; and 2). In thepresence of cofactor, all three compounds tested (MTX, TDX and LV)dramatically inhibited the BVdUMP+rHuTS reaction. In each case, theinhibition was more pronounced than that seen in the dUMP+rHuTSreaction, or the reactions with BVdUMP in the absence of THF.

[0353] The results described above, demonstrating inhibition of theBVdUMP+TS reaction by LV, MTX and TDX, and further, that this effect ismore pronounced in the presence of cofactor (THF), suggests that NB1011activity may be modulated by other chemotherapeutics. Importantly,rescue of NB1011-treated cells is feasible by providing LV, similar tothe LV rescue from MTX. In the case of MLX, LV rescue occurs viasupplementation of intracellular folate pools, which are diminished viaMTX inhibition of dihydrofolate reductase and TS. If reduced folates arediminished within the cell during BVdUMP reaction with TS, then othercompounds that diminish intracellular thymidine or purine nucleotidepools by distinct mechanisms may give additive or synergisticanti-cellular effects when used together with NB1011. Examples of suchcompounds (Dorr and Von Hoff (1994)),include 6-mercaptopurine,thioguanine and 2i-deoxycoformycin, all of which interfere with purinemetabolism. Azacytidine-mediated inhibition of orotidylate decarboxylaseblocks pyrimidine biosynthesis, and so could lower intracellularthymidine levels in a cell by a mechanism distinct from that of NB 1011.

[0354] Pharmacogenomics of TS ECTA

[0355] 1. Comparison of TS and HER2

[0356] An important aspect of the current approach to discovery anddevelopment of novel therapeutics is the ability to identify patientswho are most likely to respond to treatment (a positive pharmacogenomicsselection). One of the pioneering drugs in this area is Herceptin, nowused to treat breast cancers which overexpress the HER2 protooncogene.Early data with anti-HER2 antibodies showed that activity on randomlyselected tumor cells and normal cells was minimal. However, if tumorcell lines were selected that had at least a 4-fold increased expressionof HER2, then a significant activity and anti-HER2 antibody could bedemonstrated, as compared to normal cells or tumor cells expressinglower amounts of the HER2 gene product (Shepard, et al. (1991) and(Lewis (1993)). The data shown in FIGS. 5A and 5B demonstrate that,similarly to the case with Herceptin.

[0357] The cell line results shown in FIG. 2 may suggest an additionalsimilarity between the TS and HER2/NEU systems. The similarity is thateach has a similar overexpression requirement (about 4-fold) whichpredicts more aggressive disease for both TS and HER2/NEU overexpressingpatients (Johnston, et al. (1994)).

[0358] 2. NB1011 is Active Against 5FU and Tomudex-resistant Colon andBreast Tumor Cell Lines

[0359] Because NB1011 has promising anticancer activity, it is importantto compare it with other chemotherapeutics with respect to safety. Theutility of NB1011 in the treatment of cancer is further strengthenedwhen it is compared with Tomudex, a chemotherapeutic which, like 5FU, isoften used to treat colon and breast cancer, among other malignancies.

[0360] The results (FIG. 10) show that while NB1011 is more than 10-foldless toxic than TDX vs. normal cells (CCD18co), it is more than 30-foldmore potent than TDX on MCF7-TDX resistant tumor cells. Similar resultshave been obtained for other TDX-resistant tumor cell lines. The lowlevel of toxicity vs. normal cells and the high activity vs. TDX^(R)tumor cells supports the application of NB1011 to drug resistant cancersthat overexpress TS.

[0361] 3. NB1011 is More Dependent Upon TS Protein Levels than TSActivity as Measured by Tritium Release from dUMP-³H

[0362] Four types of assays have been used to characterize TS levels incells and tissues. Most commonly used is the antibody-based technique(Johnston (1994) and (Johnston (1995)) but RT-PCR, 5FdUMP-binding andtritium release (van Laar (1996), van Triest (1999), Jackman (1995),Larsson (1996), Komaki (1995) and Mulder (1994)) have also been measuredin various studies. For characterization of cell lines we have focusedon Western blotting and tritium release from ³H-dUMP. These assays werechosen because antibody-detection is commonly used for clinical samplesand tritium release from labeled deoxyuridine is a direct measure of TScatalytic activity in cells.

[0363] Cells were grown and characterized as described above. TSexpression level is relative to CCD18co, a normal colon epithelial cellline. Tritium release is background substracted as described in Methods.ND=Not detectable above background.

[0364] Analysis of the data presented in Table 7 indicates that there isa closer relationship between TS protein level and sensitivity to NB1011than between TS activity (tritium release from ³H-dUMP) and NB1011sensitivity. In each set of matched parental and drug-resistant tumorcell types, the drug-resistant derivatives, each with more TS proteinthan the parent, also have an increased sensitivity to NB1011. However,when the same comparison is done with respect to TS activity, theparental cell lines often have comparable, or greater, TS activity andare less sensitive to NB1011 -mediated cytotoxicity.

[0365] While these results could occur via a number of differentmechanisms, or combinations of mechanisms, it is likely that ³H-dUMPconversion to dTMP (and accompanying tritium release) may be subject tolimitation by some component, perhaps cofactor availability. However,since conversion of BVdUMP is not dependent upon cofactor, then itsreaction with TS can continue even in a cellular milieu in whichcofactor is limiting. This observation is important because TSsubstrates as therapeutics would not be attempted based upon the resultsof typical tritium release assays for TS activity in which the mostaggressive, and drug-resistant, tumor cells are observed to have a lowerTS activity than their precursors. These results lend additional supportto the proposal of selecting patients for TS ECTA therapy based simplyon the level of TS detected by antibody staining.

[0366] 4. TS Levels in Tumor Samples Often Exceed a 4-fold Increase OverNormal Tissue

[0367] The results shown above suggest that TS ECTA therapy, at leastwith NB1011, will be most effective when used in patients whose cancersoverexpress TS at least four-fold.

[0368] The literature (Johnston (1994), Bathe (1999), Leichman (1998)and Lenz (1995)) suggests that overexpression in the range of 4-foldoccurs in about 50% of cancers, and furthermore, that this level ofoverexpression predicts a more aggressive disease. To confirm thefrequency of at least 4-fold overexpression of TS in human colon cancer,we obtained matched normal and tumor samples from the Cooperative HumanTissue Network. These samples were analyzed for TS mRNA level viaRT-PCR, which gives results comparable to immunohistochemistry(Johnston, et al. (1994)). The results of the RT-PCR evaluation of thesamples is shown in FIG. 9.

[0369] Five of the seven samples analyzed above have at least a 4-foldlevel of overexpressed TS as determined by the RT-PCR assay. None ofthese patients were previously treated with chemotherapy, which suggeststhat this frequency and level of overexpression is associated withinvasive disease and not due to selection by chemotherapy. It isexpected that cancer cells that have been exposed to TS inhibitors suchas Tomudex or the anabolic derivative of 5FU or 5-FdUrd, 5FdUMP, may beselected for increased expression (Lonn, et al. (1996)). The averagedegree of overexpression, as measured by RT-PCR for all 7 samples, isabout 4.7-fold. These data suggest that greater than 4-foldoverexpression of TS in tumor foci is a common event.

[0370] 5. Experimental Therapy of 5FU-resistant Human Colon Cancer

[0371] The most important diseases for new compounds that target TS arethe gastrointestinal cancers. To study the activity of NB1011 in an invivo model, H630R10, 5FU-resistant human colon cancer cells, were grownsubcutaneously to an average tumor size of 50 mm³ in nude mice. The micewere then treated, with excipient (DMSO, 5FU or NB1011).

[0372] Doses of 3.5 mg, 2.5 mg, and 1.25 mg of NB1011 were administereddaily for 5 days, either peritumorally or intraperitoneally totumor-bearing mice. FIG. 8A shows the initial block in tumor growthinduced by treatment for 5 days with NB1011, as compared to excipient or5FU treated animals. Although no statistically significant dose responserelationship is evident among the NB1011 groups, there is a significantdifference between the NB1011 groups vs. either the 5FU or excipientcontrols, starting with Day 6. This difference is maintained (FIG. 9B)until the control animals were sacrificed at Day 25, even though therapywas discontinued after Day 5. TABLE 3 Comparison of Kinetic Parametersof Bacterial and rHuTS Kinetic Constants Lactobacillus casei rHuTS dumpK_(m) 3.0 μM 7.7 μM K_(cat) 6.4 s⁻¹ 0.2 s⁻¹ K_(cat/)K_(m) 2.1 × 10⁶ M⁻¹s⁻¹ 2.6 × 10⁴ M⁻¹ s⁻¹ K_(l) (of BVdUMP) 0.6 μM 4.5 μM BVdUMP K_(m) 3.3μM 16 μM K_(cat) 0.018 s⁻¹ 0.0067 s⁻¹ K_(cat/)K_(m) 5.6 × 10³ M⁻¹ s⁻¹4.2 × 10³ M⁻¹s⁻¹ K_(l) (of dUMP) 2.0 μM 17.5 μM Relative catalytic385-fold 60-fold efficiency (dUMP vs BVdUMP)

[0373] TABLE 4 Inhibition of rHuTS reactions by Tomudex and 5-FdUMPTomusdex Substrate + Cofactor No Inhibitor (500 nM) 5-FdUMP (500 nM)BVdUMP + THF 109 ± 16 67 ± 3 44 ± 2 RFU/min (61%)  (40%) (100%) BVdUMP −THF  75 ± 11 34 ± 3  93 ± 13 (100%) (45%) (129%)   dUMP + THF 1500 ± 20690 ± 40 290 ± 70 nmoles/min (46%)  (19%) (100%)

[0374] TABLE 5 Cytotoxicity of NB1011 vs. 5FU on Normal and Tumor CellStrains IC₅₀ (μM) IC₅₀ (μM) Normal Cells NB101.1 5FU Tumor Cells NB101.15FU CCD1800 (Colon) 562 2.0 H630R10 (Colon) 65 41.6 DET551 (Skin) 2620.8 HT1080 (Colon) 449 0.8 NHDF (Skin) 359 0.8 COLO320 (Colon) 401 1.5H527 (Skin) 273 1.6 COLO205 (Colon) 105 1.3 W138 (Lung) 335 1.0 SW620(Colon) 374 4.6 MRC9 (Lung) 303 1.1 SKCO1 (Colon) 184 1.4 NHLF (Lung)139 0.9 HCTC (Colon) 280 2.8 NHA (Brain) 839 0.9 MCF7 (Breast) 141 1.0NHOST (Bone) 642 4.7 MDAMB361 (Breast 365 5.0 NPRSC (Prostate) 369 1.7MDAMB468 (Breast) 172 4.4 NHEPF (Liver) 2085 1.7 SW527 (Breast) 431 4.3Average 561 1.6 NCI H520 (Lung) 135 0.6 SKLU1 (Lung) 270 7.9 SOAS2(Bone) 232 1.4 PANC1 (Pancreas) 492 1.9 SKOV3 (Ovary) 484 3.0 PC3(Prostate) 184 0.9 HEPG2 (Liver) 704 22.8 SKHEP1 (Liver) 247 1.7 A431(Skin) 266 0.2 MCIxc (Brain) 61. 1.2 Average 288 5.3 NB101.1 5FUTherapeutic index (N/T) 1.95 0.30

[0375] TABLE 6 NB1011 cytotoxicity on cell lines engineered to expressHuTS. IC₅₀ TS Level NB1011 FUDR 5-FU TDX Cell Line (%)* (μM) (μM) (μM)(μM)     C/HT1080 100 320 <0.1 1.0 3.6 TSL/HT1080 409 196 2.2 1.7 24TSL/HT1080 702 0.8 3.1 3.5 153

[0376] TABLE 7 Tomudex Inhibits NB1011 Mediated Cytotoxicity [Tomudex](nM) 0 nM 1 nM 10 nM 100 nM 1000 nM NB1011IC₅₀ 5.7 25.5 87.7 140.3 103.0(μM) Fold Protection 1 4.5× 15.4× 24.6× 18.1×

[0377] TABLE 8 Impact of Folate Inhibitors BVdUMP, Inhibitor with THFBVdUMP, w/o THF dUMP, with THF None 100% 138% 100% MTX  10%  24%  31% LV 17%  97%  77% TDX  0%  25%  18%

[0378] TABLE 9 NB1011 activity is more associated with TS protein thanwith tritium release Drug Tritium NB1011- Cell Line Selection TS ProteinRelease IC₅₀ H630 None 288 3206 414 Colon cancer 5FU 2350 1840 65 TDX671 3980 2.3 RKO None 142 4920 136 Colon cancer TDX 279 1625 28 MCF7None 178 5185 327 Breast cancer TDX 1980 875 2.8 N1S1 None 197 12,565494 5FU 1241 ND 204

[0379] TABLE 10 MDF7 TDX cells selected for resistance to NB1011 aremore sensitive to 5-Fluorouracil and Tomudex IC₅₀ (micromolar)* RelativeTS 5-FU Tomudex NB1011 Protein Level MCF7 10− .026− 291−  1X− MCF7 TDX32  >10 2 11X MCF7 TDX/1011 2 .041 240 4X

EXAMPLE 19 Co-Administration

[0380] Cell lines: Normal human colon epithelial cells (CCD18co) andskin fibroblasts (Det55 1) were purchased from ATCC (Rockville,Md.).MCF7TDX, human breast carcinoma cells resistant to 2 μM Tomudex wereobtained from Dr. Patrick Johnston, Queens University, Belfast. H630R10,human colorectal carcinoma cells resistant to 10 μM 5-Fluorouracil wereobtained from Dr. Edward Chu (Yale Cancer Center) and Dr. Dennis Slamon(UCLA). The MCF7TDX and the H630R10 cell lines have been previouslydescribed in Drake, J. C. et al. (1996) and Copur, S. et al. (1995)respectively.

[0381] Chemicals: Dipyridamole and nitrobenzylthioinosine were purchasedfrom ICN Biomedicals (Aurora, Ohio). 5-Fluorouracil was purchased fromSigma (St. Louis, MO). Tomudex was provided by Zeneca (Wilmington,Del.).

[0382] Culture Conditions: All cells were cultured under standardconditions of 37° C., 95% humidified air, 5% CO₂ in RPMI 1640 culturemedium containing 10% fetal calf serum (Life Technologies) andpenicillin/streptomycin/fungizone. MCF7TDX cells were maintainedcontinuously in 2 μM Tomudex, and H630R10 cells were maintainedcontinuously in 10 μM 5-FU. The medium was renewed or the cells werepassaged about every three days to maintain optimal growth conditions.Normal cells were passaged a maximum of 15 times to avoid senescence.

[0383] Cytotoxicity Studies: 384-well interaction screening assay. 500cells per well were transferred to a 384-well tissue culture plate(Corning Inc., Corning, N.Y.) and allowed to attach for 24 hours instandard culture conditions. Compounds were then applied in abidirectional (checker board) pattern (Chou, T. C. and Talalay, P.(1984)). Following a 5-day incubation, the redox indicator dye,alamarBlue (AccuMed International, Westlake, Ohio) was added to eachwell at a 10% v/v ratio, and fluorescence was monitored at 535excitation, 595 emission. Cytotoxic effect levels and drug interactionswere assessed by the combination index method (Chou, T. C. and Talalay,P. (1984) and Bible, K. C. et al. (1997)), described briefly below.96-well combination cytotoxicity assay. Exponentially growing cells weretransferred at a density of 1.0-5.5×10³ cells per well to a 96-welltissue culture plate and allowed to attach for 24 hours. Compounds werethen applied in duplicate half log serial dilutions. Each compound wastested separately, and mixed together at a single molar ratioapproximately equal to the ratio of the individual IC₅₀ values. After anadditional 72 hour incubation, cells were washed once with PBS andstained with 0.5% crystal violet in methanol. Plates were washed gentlyin water to remove unbound stain and allowed to dry overnight. Crystalviolet stain bound to the total protein of attached cells wasredissolved in Sorenson's buffer (0.025M sodium citrate, 0.025M citricacid in 50% ethanol), and absorbance monitored at 535 nM. Sigmoid curveswere fit according to the Hill inhibitory Emax model, and IC₅₀calculated as the average of three or more separate determinations.Where applicable, the combination index for multiple drug effects wascalculated according to the median-effect principle (Chou, T. C. andTalalay, P. (1984)) using the CalcuSyn software from Biosoft (Ferguson,Mo.). Briefly, the ICso and the slope parameter (m) for each agent alonewere determined from the median effect plot, an x,y plot of log(D) vslog (f_(a)/f_(u)) based on Chou's median effect equation:

f _(a) /f _(u)=(D/D _(m))^(m)  [Equation 1]

[0384] where D=dose of the drug, D_(m)=IC₅₀ as determined from thex-intercept of the median effect plot, f_(a)=fraction of cells affected,f_(u)=fraction of cells unaffected (f_(u) =1−f_(a)), and m=an exponentsignifying the steepness of the sigmoid dose-effect curve. Onlyexperiments with linear correlation coefficients (r) >0.9 were acceptedfor analysis. A combination index (CI) was then calculated to assesssynergism or antagonism according to the following equation whichassumes an independent mechanism of drug action (mutual exclusivity):

CI=(D)₁/(D _(x))₁+(D)₂/(_(D) _(x))₂+(D)₁(D)₂/(D _(x))₁ (D_(x))₂  [Equation 2]

[0385] where (D)₁ and (D)₂ are the concentrations of drug 1 and drug 2which combined produce x% inhibition, and (D_(x))₁ and (D_(x))₂ are theconcentrations of each drug which alone produce x% inhibition. CI=1indicates an additive interaction, CI<1 indicates synergy, and CI>1indicates antagonism. For each experiment Cl's from several differenteffect levels and concentrations of a constant molar ratio wereaveraged. Student t-tests were applied to determine if the averagediffered significantly from 1.

[0386] Results:

[0387] 384-well screening studies. To identify drugs which potentiallysynergize with NB1011, combination cytotoxicity experiments wereperformed with NB1011 and each of 10 antitumor agents from severaldifferent mechanistic classes using MCF7TDX and H630R10 tumor cells.Results from these initial 384-well alamarBlue screening assays areshown in Table 11. In general, a combination index of <1 indicatessynergy, ˜1 indicates additivity, and >1 indicates antagonism (Pegram,M. D. et al. (1999)). TABLE 11 Drugs screened for interaction withNB1011 Combination Index ± s.e.m. Drug Class MCF7 TDX H630R10 IrinotecanInhibition of topoisomerase I 1.36 ± 0.38 1.26 ± 0.20 topotecan 2.45 ±0.85 ND Etoposide Inhibition of topoisomerase II 3.13 ± 0.58 1.96 ± 0.28Vinblastine Inhibition of microtubule 1.09 ± 0.16 0.78 ± 0.32 assemblyTaxol Stabilization of microtubules 1.41 ± 0.32 0.99 ± 0.15 CisplatinDNA damage 1.51 ± 0.35 ND Thiotepa Alkylation 2.23 ± 0.45 ND DoxorubicinInhibition of nucleic acid 0.55 ± 0.06 1.05 ± 0.13 synthesis 5-fluoro-Inhibition of TS, DNA/RNA 3.19 ± 0.35 ND uracil incorporationMethotrexate Antifolate, inhibition of 1.78 ± 0.44 ND DHFR, TS

[0388] Two of the ten agents screened, vinblastine and doxorubicin,showed potential synergy (CI≦1.1) with NB1011 in MCF7TDX and H630R10cell. Two of the remaining 8 agents, irinotecan and taxol showed anadditive or antagonistic interaction (CI=1-1.4) with NB1011, while allthe other agents showed antagonism (CI>1.5). The most antagonisticinteraction was observed with 5-Fluorouracil which gave CI=3.19 inMCF7TDX cells. In light of these results, vinblastine and doxorubicinwere chosen for further study using a 96-well crystal violet combinationcytotoxicity assay. 96-well combination cytotoxicity studies. The96-well format was chosen for more detailed drug interaction studies.Three additional agents were included in the 96-well assay: oxaliplatin,a new platinum analog DNA damaging agent; dipyridamole (DP) andp-nitrobenzylthioinosine (NBMPR), both potent inhibitors ofequilibrative nucleoside transport processes. Oxaliplatin was tested toconfirm the antagonism results for cisplatin. The nucleoside transportinhibitors were tested because published data (Tsavaris, N. etal.(1990), Grem, J. L. (1992) and Wright, A. M. et al. (2000)) suggestedthey may modulate the activity of nucleoside based drugs. To analyzewhether any of these drugs would enhance the activity of NB1011specifically in tumor cells, two normal cell types, Det551 and CCD18co,were included in the assays. Results of these experiments are shown inTable 12. TABLE 12 Average combination index (CI) values for drugstested in combination with NB1011 in tumor and normal cells P MolarNB1011 Drug Dose Inter- Drug Cell Line CI ±SEM value Ratio^(a) Dose (μM)(μM) action^(b) Dipyridamole H630R10 0.75 0.11 0.052 2  11-150 5.5-75 Syn MCF7TDX 0.51 0.06 0.001 0.2 1.1-3.2 5.5-16  Syn Det551 1.17 0.230.484 5  5.8-375  1.2-75  Add CCD18co 1.30 0.08 0.008 5  81-375 16-75Ant p-Nitrobenzyl- H630R10 0.35 0.07 0.001 1  1.5-500   1.5-500  SynThioinosine MCF7TDX 0.57 0.17 0.029 3.33 0.15-150  0.045-45   Syn(NBMPR) Det551 1.43 0.16 0.026 3.33  32-300 9.7-90  Ant CCD18co 3.931.00 0.019 3.33  32-300 9.7-90  Ant Vinblastine H630R10 0.63 0.10 0.0036000 4.1-54  0.0005-0.015  Syn MCF7TDX 1.44 0.29 0.186 2000 0.4-1.90.0005-0.015  Ant Det551 0.54 0.10 0.003 50000 2.9-47  0.0005-0.015 SynCCD18co 0.65 0.10 0.008 50000  17-135 0.0005-0.015  Syn OxaliplatinH630R10 1.78 0.06 0.001 120  6.9-150  0.1-1.3 Ant MCF7TDX 2.24 0.330.004 12 0.6-15  0.1-1.3 Ant Doxorubicin H630R10 1.39 0.13 0.012 300117-150 0.039-0.5  Ant MCF7TDX 1.96 0.25 0.004 600 1.9-15  0.001-0.025Ant

[0389] As can be seen in Table 12, doxorubicin, although promising inthe initial screening assay, failed to synergize in the more detailed 96well cytotoxicity assay (CI =1.39 and 1.96 in H630R10 and MCF7TDX cells,respectively). Oxaliplatin had an antagonistic interaction in the tumorcells (CI=1.78 and 2.24, respectively). Since both oxaliplatin anddoxorubicin antagonized NB1011 in the tumor cells, they were not testedin the normal cell assays. Consistent with the initial screening data,vinblastine synergized with NB1011 in H630R10 cells (CI=0.63), howeverit antagonized NB1011 in MCF7TDX cells (CI=1.44). Furthermore, in Det551and CCD18co normal cells, vinblastine interacted synergistically withNB1011 to a similar extent as in H630R10 cells (CI=0.54 and 0.65,respectively). This lack of selectivity in the potentiation of NB1011 byvinblastine would most likely limit the use of this combination in theclinic. The nucleoside transport inhibitor, dipyridamole, synergizedwith NB1011 in the tumor cells (CI=0.75 and 0.51), but failed tosynergize with NB1011 in the normal cells (CI=1.17 and 1.30). Similarly,NBMPR, another NT inhibitor, showed synergy with NB31011 in the tumorcells (CI=0.35 and 0.57), but produced no synergy in the normal cells(CI=1.43 and 3.93). Taken together this data indicate that 2 of the 13agents tested, DP and NBMPR, which are both inhibitors of equilibrativenucleoside transport, potentiate the activity of NB1011. Thisenhancement of NB1011 activity by DP and NBMPR appears specific for thetumor cells tested, since no synergy was observed for these combinationsin the two normal cell types analyzed.

EXAMPLE 20

[0390] Induction and Assessment of Arthritis

[0391] Arthritis was induced in male DBA/1 mice (8-10 weeks old) byintradermal injection of bovine type II collagen, purified in-house atthe Kennedy Institute of Rheumatology as previously described (Miller,E. J. et al. (1972)). Collagen was administered in complete Freund'sadjuvant (Difco, Detroit, Mich.). Onset of arthritis is expected to bevariable. Arthritis onset is considered to occur on the day thatswelling and/or erythema were observed. Clinical score is a composite ofdisease severity and the number of limbs affected, and is monitoreddaily from onset of disease and used as an assessment of diseaseprogress. An example for scoring is: 0, Normal; 1, slight swelling witherythema; 2, pronounced swelling; 3, joint rigidity. In addition, theextent of paw swelling reflects the degree of edema in affected limbs.

[0392] Anti-TNF antibody used in these experiments was as described byMarinova-Mutafchieva, L. et al. (2000). NB1011 was administered daily byintraperitoneal administration at 2.5 mg total dose per day. Anti-TNFantibody was compared with NB1011 because, at present, antiTNF antibodyis the optimal single agent for treatment of collagen induced arthritis(Marinova-Mutafchieva, L. et al. (2000)).

[0393] Success in this model has been shown to be predictive forclinical success in the development of new agents to treat inflammatorydisease, especially rheumatoid arthritis (Elliott et al. (1994) andFeldmann et al. (1998)). This model therefore represents an idealsetting for establishing proof of concept for new agents to treatrheumatoid arthritis, and potentially other autoimmune and inflammatorydiseases.

[0394] Following immunization with collagen, mice were maintained untila significant clinical score for disease progression was achieved(between 2.5 and 3.5). Mice were then treated with control salineinjections, NB1011, or with anti-TNF antibody as a positive control. Theresults showed that the NB1011-treated group exhibited significantdisease suppression (p<0.05), similar to the anti-TNF control, whencompared with the saline-treated control group. There was no significantdifference between the NB1011 and anti-TNF groups with regard toclinical score. Paw swelling is an alternative measure of CIA diseaseseverity. When paw swelling was used as a criteria for diseasesuppression, comparable results were observed. In this second measure ofefficacy, both the NB1011 and anti-TNF groups demonstrated significantdisease suppression as compared to the saline-treated control group(p<0.05). Again, there was no significant difference between the NB1011and anti-TNF groups, although suppression of swelling may have been lessdramatic with NB1011. A further significant outcome of this work is thatby comparison with earlier reported work, NB1011 appears to haveactivity superior to anti-angiogenesis agents, an anti-CD4immunosuppressive agent, and cannabidiol, a third experimental agentcurrently being considered for use to treat rheumatoid arthritis, andpotentially other autoimmune and inflammatory disorders (Malfait, A. M.et al. (2000); Miotla, J. et al. (2000); Marinova-Mutafchieva, L. et al.(2000)).

[0395] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications will be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

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What is claimed is:
 1. A compound having the formula:

wherein R₁ is selected from the group consisting of H, alkyl, alkenyl,alkynyl, vinyl, propargyl and substituted derivatives thereof; whereinR₂ and R₃ are independently the same or different and are selected fromthe group consisting of Br, Cl, F, I, H, OH, OC(═O)CH₃, —O-and —O—Rg,wherein Rg is a hydroxyl protecting group other than acetyl; wherein R₈is a side chain of any naturally occurring amino acid, its analogue orits isomer; and wherein R₉ is selected from the group consisting ofhydrogen, an aliphatic group, an alicyclic group, an aromatic group, aheterocyclic group and an adamantly group and derivatives and analogsthereof; and any enantiomeric, diasteriomeric, or stereoisomeric form,including D-form, L-form, α-anomeric form, and β-anomeric form, and itspharmaceutically acceptable salts.
 2. The compound of claim 1, whereinR₈ is the side chain of a naturally occurring amino acid, its analogueor isomer, selected from the group consisting of alanine, glycine,tryptophan, leucine and aspartic acid.
 3. The compound of claim 1,wherein R₁ is a halogen-substituted vinyl derivative.
 4. The compound ofclaim 3, wherein R₁ is bromovinyl.
 5. The compound of claim 4, whereinR₈ is alanine, with the proviso that R₉ is not methyl.
 6. The compoundof claim 5, wherein R₉ is selected from the group consisting of—CH₂-phenyl, —CH₃, —CH₂-cyclopropyl, cyclohexyl, iso-propyl,—CH₂-tert-butyl, cycloheptyl, cyclooctyl and —CH₂-adamantyl.
 7. Thecompound of claim 1, wherein the compound has the formula:


8. The compound of claim 1, wherein the compound has the formula:


9. The compound of claim 1, wherein the compound has the formula:


10. The compound of claim 1, wherein the compound has the formula:


11. The compound of claim 1, wherein the compound has the formula:


12. The compound of claim 1, wherein the compound has the formula:


13. The compound of claim 1, wherein the compound has the formula:


14. The compound of claim 1, wherein the compound has the formula:


15. The compound of claim 1, wherein the compound has the formula:


16. The compound of claim 1, wherein the compound has the formula:


17. The compound of claim 1, wherein the compound has the formula:


18. The compound of claim 1, wherein the compound has the formula:


19. The compound of claim 1, wherein the compound has the formula:


20. The compound of claim 1, wherein the compound has the formula:


21. The compound of claim 1, wherein the compound has the formula:


22. The compound of claim 1, wherein the compound has the formula:


23. The compound of claim 1, wherein the compound has the formula:


24. The compound of claim 1, wherein the compound has the formula:


25. The compound of claim 1, wherein the compound has the formula:


26. The compound of claim 1, wherein the compound has the formula:


27. A composition comprising the compound of claim 1 and a carrier. 28.A pharmaceutical composition comprising the compound of claim 1 and apharmaceutically acceptable carrier.
 29. A method of inhibiting theproliferation of a cell selected from the group consisting of a cancercell or a cell infected with an infectious agent, comprising contactingthe cell with an effective amount of the compound of claim
 1. 30. Amethod for treating a cell or tissue related to an autoimmune disorderor inflammatory condition, comprising contacting the cell or tissue withan effective amount of the compound of claim 1.